Engineered biosynthetic pathways for production of histamine by fermentation

ABSTRACT

The present disclosure describes the engineering of microbial cells for fermentative production of histamine and provides novel engineered microbial cells and cultures, as well as related histamine production methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No. 62/660,875, filed Apr. 20, 2018, which is hereby incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Agreement No. HR0011-15-9-0014, awarded by DARPA. The Government has certain rights in the invention.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

This application includes a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on Apr. 17, 2019, is named ZMGNP011WO_Seq_List_ST25.txt and is 312,107 bytes in size.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to the area of engineering microbes for production of histamine by fermentation.

BACKGROUND

Biogenic amines are organic bases endowed with biological activity, which are frequently found in fermented foods and beverages. Histamine is known to exist in nature in fermented foods such as yogurt (13-36 mg/kg) [1], miso (24 mg/kg) [2], and red wine (24 mg/L) [3]. Some bacteria that live in the human gut also make histamine, and it functions to regulate the immune system by an anti-inflammatory effect [4]. Production of histamine in fermented foods relies on a source of proteins that contain histidine and microbes that histidine decarboxylase. Histamine is the decarboxylation product of histidine that is catalyzed specifically by the enzyme histidine decarboxylase (EC 4.1.1.22). Production of histamine in an industrial fermentation from simple, non-protein, carbon and nitrogen sources requires assembly of a pathway with improved biosynthesis of the amino acid precursor histidine and a highly active histidine decarboxylase.

SUMMARY

The disclosure provides engineered microbial cells, cultures of the microbial cells, and methods for the production of histamine, including the following:

Embodiment 1: An engineered microbial cell that expresses a non-native histidine decarboxylase, wherein the engineered microbial cell produces histamine.

Embodiment 2: The engineered microbial cell of embodiment 1, wherein the engineered microbial cell includes increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell.

Embodiment 3: The engineered microbial cell of embodiment 2, wherein the one or more upstream histamine pathway enzyme(s) are selected from the group consisting of an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase.

Embodiment 4: The engineered microbial cell of any one of embodiments 1-3, wherein the engineered microbial cell includes reduced activity of one or more enzyme(s) that consume one or more histamine pathway precursors, said reduced activity being reduced relative to a control cell.

Embodiment 5: The engineered microbial cell of embodiment 4, wherein the one or more enzyme(s) that consume one or more histamine pathway precursors are selected from the group consisting of an enolase, a pyruvate dehydrogenase, a pentose phosphate pathway sugar isomerase, a transaldolase, a transketolase, a ribulose-5-phosphate epimerase, and a ribulose-5-phosphate isomerase.

Embodiment 6: The engineered microbial cell of embodiment 4 or embodiment 5, wherein the reduced activity is achieved by replacing a native promoter of a gene for said one or more enzymes with a less active promoter.

Embodiment 7: The engineered microbial cell of any one of embodiments 1-6, wherein the engineered microbial cell additionally expresses a feedback-deregulated glucose-6-phosphate dehydrogenase or a feedback-deregulated ATP phosphoribosyltransferase.

Embodiment 8: An engineered microbial cell, wherein the engineered microbial cell includes means for expressing a non-native histidine decarboxylase, wherein the engineered microbial cell produces histamine.

Embodiment 9: The engineered microbial cell of embodiment 8, wherein the engineered microbial cell includes means for increasing the activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell.

Embodiment 10: The engineered microbial cell of embodiment 9, wherein the one or more upstream histamine pathway enzyme(s) are selected from the group consisting of an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, a histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase.

Embodiment 11: The engineered microbial cell of any one of embodiments 8-10, wherein the engineered microbial cell includes means for reducing the activity of one or more enzyme(s) that consume one or more histamine pathway precursors, said reduced activity being reduced relative to a control cell.

Embodiment 12: The engineered microbial cell of embodiment 11, wherein the one or more enzyme(s) that consume one or more histamine pathway precursors are selected from the group consisting of an enolase, a pyruvate dehydrogenase, pentose phosphate pathway sugar isomerase, a transketolase, a translaldolase, a ribulose-5-phosphate epimerase, and a ribulose-5-phosphate isomerase.

Embodiment 13: The engineered microbial cell of embodiment 11 or embodiment 12, wherein the reduced activity is achieved by means for replacing a native promoter of a gene for said one or more enzymes with a less active promoter.

Embodiment 14: The engineered microbial cell of any one of embodiments 8-13, wherein the engineered microbial cell additionally includes means for expressing glucose-6-phosphate dehydrogenase or a feedback-deregulated ATP phosphoribosyltransferase.

Embodiment 15: The engineered microbial cell of any one of embodiments 1-14, wherein the engineered microbial cell includes a fungal cell.

Embodiment 16: The engineered microbial cell of embodiment 15, wherein the engineered microbial cell includes a yeast cell.

Embodiment 17: The engineered microbial cell of embodiment 16, wherein the yeast cell is a cell of the genus Saccharomyces or Yarrowia.

Embodiment 18: The engineered microbial cell of embodiment 17, wherein the yeast cell is a cell of the genus Saccharomyces and of the species cerevisiae.

Embodiment 19: The engineered microbial cell of embodiment 17, wherein the yeast cell is a cell of the genus Yarrowia and of the species lipolytica.

Embodiment 20: The engineered microbial cell of any one of embodiments 1-19, wherein the non-native histidine decarboxylase includes a histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 or from Acinetobacter baumannii strain AB0057.

Embodiment 21: The engineered microbial cell of any one of embodiments 1 and 16-20, wherein the engineered microbial cell includes increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell, wherein the one or more upstream histamine pathway enzyme(s) comprise an ATP phosphoribosyltransferase.

Embodiment 22: The engineered microbial cell of embodiment 21 wherein the increased activity of the ATP phosphoribosyltransferase is achieved by heterologously expressing it.

Embodiment 23: The engineered microbial cell of embodiment 22, wherein the heterologous ATP phosphoribosyltransferase has at least 70% amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae.

Embodiment 24: The engineered microbial cell of any one of embodiments 16-23, wherein the engineered microbial cell includes a feedback-deregulated variant of a Corynebacterium glutamicum ATP phosphoribosyltransferase.

Embodiment 25: The engineered microbial cell of any one of embodiments 1-14, wherein the engineered microbial cell is a bacterial cell.

Embodiment 26: The engineered microbial cell of embodiment 25, wherein the bacterial cell is a cell of the genus Corynebacteria or Bacillus.

Embodiment 27: The engineered microbial cell of embodiment 26, wherein the bacterial cell is a cell of the genus Corynebacteria and of the species glutamicum.

Embodiment 28: The engineered microbial cell of embodiment 26, wherein the bacterial cell is a cell of the genus Bacillus and of the species subtilis.

Embodiment 29: The engineered microbial cell of any one of embodiments 25-28, wherein the non-native histidine decarboxylase includes a histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Acinetobacter baumannii or from Lactobacillus sp. (strain 30a).

Embodiment 30: The engineered microbial cell of any one of embodiments 1 and 25-29, wherein the engineered microbial cell includes increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell, wherein the one or more upstream histamine pathway enzyme(s) comprise an ATP phosphoribosyltransferase and an imidazole-glycerol phosphate dehydratase.

Embodiment 31: The engineered microbial cell of embodiment 30, wherein the increased activity of the ATP phosphoribosyltransferase or the imidazole-glycerol phosphate dehydratase is achieved by heterologously expressing it.

Embodiment 32: The engineered microbial cell of embodiment 31, wherein the heterologous ATP phosphoribosyltransferase has at least 70% amino acid sequence identity with an ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c or from Salmonella typhimurium LT2, or the heterologous imidazole-glycerol phosphate dehydratase has at least 70% amino acid sequence identity with an imidazole-glycerol phosphate dehydratase from Corynebacterium glutamicum.

Embodiment 33: The engineered microbial cell of any one of embodiments 25-32, wherein the engineered microbial cell includes a feedback-deregulated variant of a Salmonella typhimurium ATP phosphoribosyltransferase.

Embodiment 34: The engineered microbial cell of any one of embodiments 1-33, wherein, when cultured, the engineered microbial cell produces histamine at a level of at least 20 mg/L of culture medium.

Embodiment 35: The engineered microbial cell of embodiment 34, wherein, when cultured, the engineered microbial cell produces histamine at a level of at least 300 mg/L of culture medium.

Embodiment 36: A culture of engineered microbial cells according to any one of embodiments 1-35.

Embodiment 37: The culture of embodiment 36, wherein the engineered microbial cells are present in a concentration such that the culture has an optical density at 600 nm of 10-500.

Embodiment 38: The culture of any one of embodiments 36-37, wherein the culture includes histamine.

Embodiment 39: The culture of any one of embodiments 36-38, wherein the culture includes histamine at a level at least 20 mg/L of culture medium.

Embodiment 40: A method of culturing engineered microbial cells according to any one of embodiments 1-35, the method including culturing the cells under conditions suitable for producing histamine.

Embodiment 41: The method of embodiment 40, wherein the method includes fed-batch culture, with an initial glucose level in the range of 1-100 g/L, followed controlled sugar feeding.

Embodiment 42: The method of any one of embodiments 40-41, wherein the fermentation substrate includes glucose and a nitrogen source selected from the group consisting of urea, an ammonium salt, ammonia, and any combination thereof.

Embodiment 43: The method of any one of embodiments 40-42, wherein the culture is pH-controlled during culturing.

Embodiment 44: The method of any one of embodiments 40-43, wherein the culture is aerated during culturing.

Embodiment 45: The method of any one of embodiments 40-44, wherein the engineered microbial cells produce histamine at a level at least 20 mg/L of culture medium.

Embodiment 46: The method of any one of embodiments 40-45, wherein the method additionally includes recovering histamine from the culture.

Embodiment 47: A method for preparing histamine using microbial cells engineered to produce histamine, the method including: (a) expressing a non-native histidine decarboxylase in microbial cells; (b) cultivating the microbial cells in a suitable culture medium under conditions that permit the microbial cells to produce histamine, wherein the histamine is released into the culture medium; and isolating histamine from the culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Biosynthetic pathway for histamine.

FIG. 2: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Corynebacteria glutamicum. (See also Example 1, Table 1.)

FIG. 3: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Saccharomyces cerevisiae. (See also Example 1, Table 1.)

FIG. 4: Histamine titers measured in the extracellular broth following fermentation by the second-round engineered host Corynebacteria glutamicum. (See also Example 1, Table 2.)

FIG. 5: Histamine titers measured in the extracellular broth following fermentation by the second-round engineered host Saccharomyces cerevisiae. (See also Example 1, Table 2.)

FIG. 6: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Yarrowia lipolytica. (See also Example 2, Table 4.)

FIG. 7: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Bacillus subtilis.

FIG. 8: Histamine acid titers measured in the extracellular broth following fermentation of Saccharomyces cerevisiae expressing the host evaluation designs.

FIG. 9: Histamine acid titers measured in the extracellular broth following fermentation of Corynebacteria glutamicum expressing the host evaluation designs.

FIG. 10: Histamine titers measured in the extracellular broth following fermentation by the third-round engineered host Saccharomyces cerevisiae. (Improvement round.)

FIG. 11: Integration of Promoter-Gene-Terminator into Saccharomyces cerevisiae and Yarrowia lipolytica.

FIG. 12: Promoter replacement in Saccharomyces cerevisiae and Yarrowia lipolytica.

FIG. 13: Targeted gene deletion in Saccharomyces cerevisiae and Yarrowia lipolytica.

FIG. 14: Integration of Promoter-Gene-Terminator into Corynebacteria glutamicum and Bacillus subtilis.

DETAILED DESCRIPTION

This disclosure describes a method for the production of the small molecule histamine via fermentation by a microbial host from simple carbon and nitrogen sources, such as glucose and urea, respectively. This objective can be achieved by introducing a non-native metabolic pathway into a suitable microbial host for industrial fermentation of large-scale chemical products. Illustrative hosts include Saccharomyces cerevisiae, Yarrowia lypolytica, Corynebacteria glutamicum, and Bacillus subtilis. The engineered metabolic pathway links the central metabolism of the host to a non-native pathway to enable the production of histamine. The simplest embodiment of this approach is the expression of an enzyme, a non-native histidine decarboxylase enzyme, in a microbial host strain that can produce histidine. Further engineering of the metabolic pathway by modification of the microbial host central metabolism through overexpression and mutation of a key upstream pathway enzyme, ATP phosphoribosyltransferase, enabled titers of 505 mg/L histamine to be achieved.

The following disclosure describes how to engineer a microbe with the necessary characteristics to produce industrially feasible titers of histamine from simple carbon and nitrogen sources. Active histidine decarboxylases have been identified, and it has been found that feedback-deregulated ATP phosphoribosyltransferase and/or constitutive expression of native ATP phosphoribosyltransferase improve the titers of histidine by fermentation.

Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

The term “fermentation” is used herein to refer to a process whereby a microbial cell converts one or more substrate(s) into a desired product (such as histamine) by means of one or more biological conversion steps, without the need for any chemical conversion step.

The term “engineered” is used herein, with reference to a cell, to indicate that the cell contains at least one targeted genetic alteration introduced by man that distinguishes the engineered cell from the naturally occurring cell.

The term “native” is used herein to refer to a cellular component, such as a polynucleotide or polypeptide, that is naturally present in a particular cell. A native polynucleotide or polypeptide is endogenous to the cell.

When used with reference to a polynucleotide or polypeptide, the term “non-native” refers to a polynucleotide or polypeptide that is not naturally present in a particular cell.

When used with reference to the context in which a gene is expressed, the term “non-native” refers to a gene expressed in any context other than the genomic and cellular context in which it is naturally expressed. A gene expressed in a non-native manner may have the same nucleotide sequence as the corresponding gene in a host cell, but may be expressed from a vector or from an integration point in the genome that differs from the locus of the native gene.

The term “heterologous” is used herein to describe a polynucleotide or polypeptide introduced into a host cell. This term encompasses a polynucleotide or polypeptide, respectively, derived from a different organism, species, or strain than that of the host cell. In this case, the heterologous polynucleotide or polypeptide has a sequence that is different from any sequence(s) found in the same host cell. However, the term also encompasses a polynucleotide or polypeptide that has a sequence that is the same as a sequence found in the host cell, wherein the polynucleotide or polypeptide is present in a different context than the native sequence (e.g., a heterologous polynucleotide can be linked to a different promotor and inserted into a different genomic location than that of the native sequence). “Heterologous expression” thus encompasses expression of a sequence that is non-native to the host cell, as well as expression of a sequence that is native to the host cell in a non-native context.

As used with reference to polynucleotides or polypeptides, the term “wild-type” refers to any polynucleotide having a nucleotide sequence, or polypeptide having an amino acid, sequence present in a polynucleotide or polypeptide from a naturally occurring organism, regardless of the source of the molecule; i.e., the term “wild-type” refers to sequence characteristics, regardless of whether the molecule is purified from a natural source; expressed recombinantly, followed by purification; or synthesized. The term “wild-type” is also used to denote naturally occurring cells.

A “control cell” is a cell that is otherwise identical to an engineered cell being tested, including being of the same genus and species as the engineered cell, but lacks the specific genetic modification(s) being tested in the engineered cell.

Enzymes are identified herein by the reactions they catalyze and, unless otherwise indicated, refer to any polypeptide capable of catalyzing the identified reaction. Unless otherwise indicated, enzymes may be derived from any organism and may have a native or mutated amino acid sequence. As is well known, enzymes may have multiple functions and/or multiple names, sometimes depending on the source organism from which they derive. The enzyme names used herein encompass orthologs, including enzymes that may have one or more additional functions or a different name.

The term “feedback-deregulated” is used herein with reference to an enzyme that is normally negatively regulated by a downstream product of the enzymatic pathway (i.e., feedback-inhibition) in a particular cell. In this context, a “feedback-deregulated” enzyme is a form of the enzyme that is less sensitive to feedback-inhibition than the native enzyme native to the cell. A feedback-deregulated enzyme may be produced by introducing one or more mutations into a native enzyme. Alternatively, a feedback-deregulated enzyme may simply be a heterologous, native enzyme that, when introduced into a particular microbial cell, is not as sensitive to feedback-inhibition as the native, native enzyme. In some embodiments, the feedback-deregulated enzyme shows no feedback-inhibition in the microbial cell.

The term “histamine” refers to 2-(1I-Imidazol-4-yl)ethanamine (CAS #51-45-6).

The term “sequence identity,” in the context of two or more amino acid or nucleotide sequences, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.

For sequence comparison to determine percent nucleotide or amino acid sequence identity, typically one sequence acts as a “reference sequence,” to which a “test” sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence, based on the designated program parameters. Alignment of sequences for comparison can be conducted using BLAST set to default parameters.

The term “titer,” as used herein, refers to the mass of a product (e.g., histamine) produced by a culture of microbial cells divided by the culture volume.

As used herein with respect to recovering histamine from a cell culture, “recovering” refers to separating the histamine from at least one other component of the cell culture medium.

Engineering Microbes for Histamine Production

Histamine Biosynthesis Pathway

Histamine is typically derived from the amino acid histidine. The histamine biosynthesis pathway is shown in FIG. 1. The first enzyme of the amino acid biosynthesis pathway, ATP phosphoribosyltransferase, is subject to feedback inhibition by histidine. Histamine production is enabled by the addition of a single non-native enzymatic step in Saccharomyces cerevisiae, Yarrowia lypolytica, Corynebacteria glutamicum, and Bacillus subtilis hosts, which is catalyzed by histidine decarboxylase (EC 4.1.1.22).

Engineering for Microbial Histamine Production

Any histidine decarboxylase that is active in the microbial cell being engineered may be introduced into the cell, typically by introducing and expressing the gene(s) encoding the enzyme(s)s using standard genetic engineering techniques. Suitable histidine decarboxylase may be derived from any source, including plant, archaeal, fungal, gram-positive bacterial, and gram-negative bacterial sources. Exemplary sources include, but are not limited to: Aeromonas salmonicida subsp. pectinolytica 34mel, Acinetobacter baumannii (strain AB0057), Chromobacterium haemolyticum, Chromobacterium sp. LK1, Citrobacter pasteurii, Drosophila melanogaster, Lactobacillus aviarius DSM 20655, Lactobacillus fructivorans, Lactobacillus reuteri, Lactobacillus sp. (strain 30a), Methanosarcina barkeri (strain Fusaro/DSM804), Methanosarcina barkeri str. Wiesmoor, Morganella psychrotolerans, Mus musculus, Oenococcus oeni (Leuconostoc oenos), Pseudomonas putida (Arthrobacter siderocapsulatus), Pseudomonas rhizosphaerae, Pseudomonas sp. bs2935, Solanum lycopersicum, Oryza sativa, Penicillium marneffei, Streptomyces hygroscopicus, Pseudomonas putida, Arabidopsis thaliana (Mouse-ear cress), Glycine soja (Wild soybean), Solanum lycopersicum (Tomato) (Lycopersicon esculentum), Clostridium perfringens, Lactobacillus buchneri, Drosophila melanogaster (Fruit fly), Morganella morganii (Proteus morganii), E. coli, Bos taurus (Bovine), Raoutella planticol (Klebsiella planticola), Acinetobacter baumannii, Acinetobacter haemolyticus, Photobacterium damselae, Tetragenococcus muriaticus, Moritella sp JT01, Streptococcus thermophilus, Enterobacter aerogenes, Citrobacter youngae, Raoultella omithinolytica, and Raoultella planticola.

One or more copies of histidine decarboxylase gene can be introduced into a selected microbial host cell. If more than one copy of a gene is introduced, the copies can have the same or different nucleotide sequences. In some embodiments, one or both of the heterologous gene(s) is/are expressed from a strong, constitutive promoter. In some embodiments, the heterologous histidine decarboxylase gene(s) is/are expressed from an inducible promoter. The heterologous gene(s) can optionally be codon-optimized to enhance expression in the selected microbial host cell. Illustrative codon-optimization tables for hosts used in the Examples are as follows: Bacillus subtilis Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodin.cgi?species=1423&aa=1&style=N; Yarrowia lipolytica Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodoon/cgi?species=4952&aa=1&style=N; Corynebacteria glutamicum Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodoon/cgi?species=340322&aa=1&style=N; Saccharomyces cerevisiae Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodoon/cgi?species=4932&aa=1&style=N. Also used, was a modified, combined codon usage scheme for S. cereviae and C. glutamicum, which is reproduced below.

Modified Codon Usage Table for Sc and Cg Amino Acid Codon Fraction A GCG 0.22 A GCA 0.29 A GCT 0.24 A GCC 0.25 C TGT 0.36 C TGC 0.64 D GAT 0.56 D GAC 0.44 E GAG 0.44 E GM 0.56 F TTT 0.37 F TTC 0.63 G GGG 0.08 G GGA 0.19 G GGT 0.3 G GGC 0.43 H CAT 0.32 H CAC 0.68 I ATA 0.03 I ATT 0.38 I ATC 0.59 K MG 0.6 K AAA 0.4 L TTG 0.29 L TTA 0.05 L CTG 0.29 L CTA 0.06 L CTT 0.17 L CTC 0.14 M ATG 1 N MT 0.33 N MC 0.67 P CCG 0.22 P CCA 0.35 P CCT 0.23 P CCC 0.2 Q CAG 0.61 Q CM 0.39 R AGG 0.11 R AGA 0.12 R CGG 0.09 R CGA 0.17 R CGT 0.34 R CGC 0.18 S AGT 0.08 S AGC 0.16 S TCG 0.12 S TCA 0.13 S TCT 0.17 S TCC 0.34 T ACG 0.14 T ACA 0.12 T ACT 0.2 T ACC 0.53 V GTG 0.36 V GTA 0.1 V GTT 0.26 V GTC 0.28 W TGG 1 Y TAT 0.34 Y TAC 0.66

Increasing the Activity of Upstream Enzymes

One approach to increasing histamine production in a microbial cell that is capable of such production is to increase the activity of one or more upstream enzymes in the histamine biosynthesis pathway. Upstream pathway enzymes include all enzymes involved in the conversions from a feedstock all the way to into the last native metabolite (histidine, in the illustrative microbial cells described in the Examples below). Such enzymes include an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase. Suitable upstream pathway genes encoding these enzymes may be derived from any source, including, for example, those discussed above as sources for a histidine decarboxylase gene.

In some embodiments, the activity of one or more upstream pathway enzymes is increased by modulating the expression or activity of the native enzyme(s). For example, native regulators of the expression or activity of such enzymes can be exploited to increase the activity of suitable enzymes.

Alternatively, or in addition, one or more promoters can be substituted for native promoters using, for example, a technique such as that illustrated in FIG. 12. In certain embodiments, the replacement promoter is stronger than the native promoter and/or is a constitutive promoter.

In some embodiments, the activity of one or more upstream pathway enzymes is supplemented by introducing one or more of the corresponding genes into the histidine decarboxylase-expressing microbial host cell. An introduced upstream pathway gene may be from an organism other than that of the host cell or may simply be an additional copy of a native gene. In some embodiments, one or more such genes are introduced into a microbial host cell capable of histamine production and expressed from a strong constitutive promoter and/or can optionally be codon-optimized to enhance expression in the selected microbial host cell.

Example 1 describes the successful engineering of C. glutamicum to express a heterologous histamine decarboxylase from Acinetobacter baumannii (SEQ ID NO:1) and to constitutively express a heterologous C. glutamicum imidazoleglycerol-phosphate dehydratase (SEQ ID NO:2). This strain resulted from two rounds of genetic engineering and produced histamine at a titer of 24 mg/L of culture medium. This titer was increased to 68 mg/L in a C. glutamicum strain engineered to express a histamine decarboxylase from Acinetobacter baumannii (strain AB0057) (SEQ ID NO:1) and an ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3).

Example 2 describes the successful engineering of Y. lypolytica to express a histidine decarboxylase from Acinetobacter baumannii (strain AB0057) (SEQ ID NO:1) and an ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3) to give a histamine titer of 505 mg/L. Example 2 also describes the engineering B. subtilis to express a histamine decarboxylase from Lactobacillus sp. (strain 30a) (SEQ ID NO:4) and an ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (SEQ ID NO:5) to give a histamine titer of 18 mg/L. Also in Example 2, S. cerevisiae was engineered to express a histamine decarboxylase from Chromobacterium sp. LK1 (SEQ ID NO:6) and an ATP phosphoribosyltransferase S. cerevisiae S288c (SEQ ID NO:3) to give a histamine titer of 111 mg/L.

In various embodiments, the engineering of a histamine-producing microbial cell to increase the activity of one or more upstream pathway enzymes increases the histamine titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in histamine titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30-fold, or any range bounded by any of the values listed above. (Ranges herein include their endpoints.) These increases are determined relative to the histamine titer observed in a histamine-producing microbial cell that lacks any increase in activity of upstream pathway enzymes. This reference cell may have one or more other genetic alterations aimed at increasing histamine production, e.g., the cell may express a feedback-deregulated enzyme.

In various embodiments, the histamine titers achieved by increasing the activity of one or more upstream pathway genes are at least 1, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or 10 gm/L. In various embodiments, the titer is in the range of 10 mg/L to 10 gm/L, 20 mg/L to 5 gm/L, 50 mg/L to 4 gm/L, 100 mg/L to 3 gm/L, 500 mg/L to 2 gm/L or any range bounded by any of the values listed above.

Introduction of Feedback-Deregulated Enzymes

Since histidine biosynthesis is subject to feedback inhibition, another approach to increasing histamine production in a microbial cell engineered to produce histamine is to introduce feedback-deregulated forms of one or more enzymes that are normally subject to feedback regulation. Examples of such enzymes include glucose-6-phosphate dehydrogenase and ATP phosphoribosyltransferase. A feedback-deregulated form can be a heterologous, native enzyme that is less sensitive to feedback inhibition than the native enzyme in the particular microbial host cell. Alternatively, a feedback-deregulated form can be a variant of a native or heterologous enzyme that has one or more mutations or truncations rendering it less sensitive to feedback inhibition than the corresponding native enzyme. Examples of the latter include a variant ATP phosphoribosyltransferase (from C. glutamicum) containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO:7) and a variant ATP phosphoribosyltransferase (from Salmonella typhimurium) containing the deletion of amino acids Q207 and E208 (SEQ ID NO:5).

In various embodiments, the engineering of a histamine-producing microbial cell to express a feedback-deregulated enzymes increases the histamine titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in histamine titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30-fold, or any range bounded by any of the values listed above. These increases are determined relative to the histamine titer observed in a histamine-producing microbial cell that does not express a feedback-deregulated enzyme. This reference cell may (but need not) have other genetic alterations aimed at increasing histamine production, i.e., the cell may have increased activity of an upstream pathway enzyme resulting from some means other than feedback-insensitivity.

In various embodiments, the histamine titers achieved by using a feedback-deregulated enzyme to increase flux though the histamine biosynthetic pathway are at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L, or at least 1, 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 g/L. In various embodiments, the titer is in the range of 50 μg/L to 50 g/L, 75 μg/L to 20 g/L, 100 μg/L to 10 g/L, 200 μg/L to 5 g/L, 500 μg/L to 4 g/L, 1 mg/L to 3 g/L, 500 mg/L to 2 g/L or any range bounded by any of the values listed above.

The approaches of supplementing the activity of one or more native enzymes and/or introducing one or more feedback-deregulated enzymes can be combined in histamine decarboxylase-expressing microbial cells to achieve even higher histamine production levels. For example, a histamine titer of 385 mg/L was achieved in S. cerevisiae in two rounds of engineering from the introduction of three genes: a histidine decarboxylase gene (from Chromobacterium sp. LK1) (SEQ ID NO:6), an ATP phosphoribosyltransferase (from C. glutamicum) containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO:7), and a constitutively expressed ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3). (Example 1.)

Reduction of Precursor Consumption

Another approach to increasing histamine production in a microbial cell that is capable of such production is to decrease the activity of one or more enzymes that consume one or more histamine pathway precursors. In some embodiments, the activity of one or more such enzymes is reduced by modulating the expression or activity of the native enzyme(s). Illustrative enzymes of this type include an enolase, a pyruvate dehydrogenase, a pentose phosphate pathway sugar isomerase, a transaldolase, a transketolase, a ribulose-5-phosphate epimerase, and a aribulose-5-phosphate isomerase. The activity of such enzymes can be decreased, for example, by substituting the native promoter of the corresponding gene(s) with a less active or inactive promoter or by deleting the corresponding gene(s). See FIGS. 12 and 13 for examples of schemes for promoter replacement and targeted gene deletion, respectively, in S. cervisiae and Y. lipolytica.

In various embodiments, the engineering of a histamine-producing microbial cell to reduce precursor consumption by one or more side pathways increases the histamine titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in histamine titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30-fold, or any range bounded by any of the values listed above. These increases are determined relative to the histamine titer observed in a histamine-producing microbial cell that does not include genetic alterations to reduce precursor consumption. This reference cell may (but need not) have other genetic alterations aimed at increasing histamine production, i.e., the cell may have increased activity of an upstream pathway enzyme.

In various embodiments, the histamine titers achieved by reducing precursor consumption by one or more side pathways are at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L, or at least 1, 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 g/L. In various embodiments, the titer is in the range of 50 μg/L to 50 g/L, 75 μg/L to 20 g/L, 100 μg/L to 10 g/L, 200 μg/L to 5 g/L, 500 μg/L to 4 g/L, 1 mg/L to 3 g/L, 500 mg/L to 2 g/L or any range bounded by any of the values listed above.

The approaches of increasing the activity of one or more native enzymes and/or introducing one or more feedback-deregulated enzymes and/or reducing precursor consumption by one or more side pathways can be combined to achieve even higher histamine production levels.

Microbial Host Cells

Any microbe that can be used to express introduced genes can be engineered for fermentative production of histamine as described above. In certain embodiments, the microbe is one that is naturally incapable of fermentative production of histamine. In some embodiments, the microbe is one that is readily cultured, such as, for example, a microbe known to be useful as a host cell in fermentative production of compounds of interest. Bacteria cells, including gram positive or gram negative bacteria can be engineered as described above. Examples include, in addition to C. glutamicum cells, Bacillus subtilus, B. lichenformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., P. alcaligenes, P. citrea, Lactobacilis spp. (such as L. lactis, L. plantarum), L. grayi, E. coi, E. faecium, E. gallinarum, E. casseliflavus, and/or E. faecalis cells.

There are numerous types of anaerobic cells that can be used as microbial host cells in the methods described herein. In some embodiments, the microbial cells are obligate anaerobic cells. Obligate anaerobes typically do not grow well, if at all, in conditions where oxygen is present. It is to be understood that a small amount of oxygen may be present, that is, there is some level of tolerance level that obligate anaerobes have for a low level of oxygen. Obligate anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes.

Alternatively, the microbial host cells used in the methods described herein can be facultative anaerobic cells. Facultative anaerobes can generate cellular ATP by aerobic respiration (e.g., utilization of the TCA cycle) if oxygen is present. However, facultative anaerobes can also grow in the absence of oxygen. Facultative anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes, or can be alternatively grown in the presence of greater amounts of oxygen.

In some embodiments, the microbial host cells used in the methods described herein are filamentous fungal cells. (See, e.g., Berka & Barnett, Biotechnology Advances, (1989), 7(2):127-154). Examples include Trichoderma longibrachiatum, T. viride, T. koningii, T. harzianum, Penicillium sp., Humicola insolens, H. lanuginose, H. grisea, Chrysosporium sp., C. lucknowense, Gliocladium sp., Aspergillus sp. (such as A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori), Fusarium sp. (such as F. roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora sp. (such as N. crassa or Hypocrea sp.), Mucor sp. (such as M. miehei), Rhizopus sp., and Emericella sp. cells. In particular embodiments, the fungal cell engineered as described above is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum, or F. solani. Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Patent Pub. No. 2011/0045563.

Yeasts can also be used as the microbial host cell in the methods described herein. Examples include: Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Hansenula polymorpha, Pichia stipites, Kluyveromyces marxianus, Kluyveromyces spp., Yarrowia lipolytica and Candida sp. In some embodiments, the Saccharomyces sp. is S. cerevisiae (See, e.g., Romanos et al., Yeast, (1992), 8(6):423-488). Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Pat. No. 7,659,097 and U.S. Patent Pub. No. 2011/0045563.

In some embodiments, the host cell can be an algal cell derived, e.g., from a green algae, red algae, a glaucophyte, a chlorarachniophyte, a euglenid, a chromista, or a dinoflagellate. (See, e.g., Saunders & Warmbrodt, “Gene Expression in Algae and Fungi, Including Yeast,” (1993), National Agricultural Library, Beltsville, Md.). Illustrative plasmids or plasmid components for use in algal cells include those described in U.S. Patent Pub. No. 2011/0045563.

In other embodiments, the host cell is a cyanobacterium, such as cyanobacterium classified into any of the following groups based on morphology: Chlorococcales, Pleurocapsales, Oscillatoriales, Nostocales, Synechosystic or Stigonematales (See, e.g., Lindberg et al., Metab. Eng., (2010) 12(1):70-79). Illustrative plasmids or plasmid components for use in cyanobacterial cells include those described in U.S. Patent Pub. Nos. 2010/0297749 and 2009/0282545 and in Intl. Pat. Pub. No. WO 2011/034863.

Genetic Engineering Methods

Microbial cells can be engineered for fermentative histamine production using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, see e.g., “Molecular Cloning: A Laboratory Manual,” fourth edition (Sambrook et al., 2012); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications” (R. I. Freshney, ed., 6th Edition, 2010); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction,” (Mullis et al., eds., 1994); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994).

Vectors are polynucleotide vehicles used to introduce genetic material into a cell. Vectors useful in the methods described herein can be linear or circular. Vectors can integrate into a target genome of a host cell or replicate independently in a host cell. For many applications, integrating vectors that produced stable transformants are preferred. Vectors can include, for example, an origin of replication, a multiple cloning site (MCS), and/or a selectable marker. An expression vector typically includes an expression cassette containing regulatory elements that facilitate expression of a polynucleotide sequence (often a coding sequence) in a particular host cell. Vectors include, but are not limited to, integrating vectors, prokaryotic plasmids, episomes, viral vectors, cosmids, and artificial chromosomes.

Illustrative regulatory elements that may be used in expression cassettes include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods In Enzymology 185, Academic Press, San Diego, Calif. (1990).

In some embodiments, vectors may be used to introduce systems that can carry out genome editing, such as CRISPR systems. See U.S. Patent Pub. No. 2014/0068797, published 6 Mar. 2014; see also Jinek M., et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science 337:816-21, 2012). In Type II CRISPR-Cas9 systems, Cas9 is a site-directed endonuclease, namely an enzyme that is, or can be, directed to cleave a polynucleotide at a particular target sequence using two distinct endonuclease domains (HNH and RuvC/RNase H-like domains). Cas9 can be engineered to cleave DNA at any desired site because Cas9 is directed to its cleavage site by RNA. Cas9 is therefore also described as an “RNA-guided nuclease.” More specifically, Cas9 becomes associated with one or more RNA molecules, which guide Cas9 to a specific polynucleotide target based on hybridization of at least a portion of the RNA molecule(s) to a specific sequence in the target polynucleotide. Ran, F. A., et al., (“In vivo genome editing using Staphylococcus aureus Cas9,” Nature 520(7546):186-91, 2015 Apr. 9], including all extended data) present the crRNA/tracrRNA sequences and secondary structures of eight Type II CRISPR-Cas9 systems. Cas9-like synthetic proteins are also known in the art (see U.S. Published Patent Application No. 2014-0315985, published 23 Oct. 2014).

Example 1 describes illustrative integration approaches for introducing polynucleotides and other genetic alterations into the genomes of C. glutamicum and S. cerevisiae cells.

Vectors or other polynucleotides can be introduced into microbial cells by any of a variety of standard methods, such as transformation, conjugation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE-Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. Transformants can be selected by any method known in the art. Suitable methods for selecting transformants are described in U.S. Patent Pub. Nos. 2009/0203102, 2010/0048964, and 2010/0003716, and International Publication Nos. WO 2009/076676, WO 2010/003007, and WO 2009/132220.

Engineered Microbial Cells

The above-described methods can be used to produce engineered microbial cells that produce, and in certain embodiments, overproduce, histamine. Engineered microbial cells can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more genetic alterations, such as 30-100 alterations, as compared to a native microbial cell, such as any of the microbial host cells described herein. Engineered microbial cells described in the Example below have one, two, or three genetic alterations, but those of skill in the art can, following the guidance set forth herein, design microbial cells with additional alterations. In some embodiments, the engineered microbial cells have not more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 genetic alterations, as compared to a native microbial cell. In various embodiments, microbial cells engineered for histamine production can have a number of genetic alterations falling within the any of the following illustrative ranges: 1-10, 1-9, 1-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, etc.

In some embodiments, an engineered microbial cell expresses at least one heterologous histamine decarboxylase, such as in the case of a microbial host cell that does not naturally produce histamine. In various embodiments, the microbial cell can include and express, for example: (1) a single heterologous histamine decarboxylase gene, (2) two or more heterologous histamine decarboxylase genes, which can be the same or different (in other words, multiple copies of the same heterologous histamine decarboxylase genes can be introduced or multiple, different heterologous histamine decarboxylase genes can be introduced), (3) a single heterologous histamine decarboxylase gene that is not native to the cell and one or more additional copies of an native histamine decarboxylase gene, or (4) two or more non-native histamine decarboxylase genes, which can be the same or different, and one or more additional copies of an native histamine decarboxylase gene.

This engineered host cell can include at least one additional genetic alteration that increases flux through the pathway leading to the production of histidine (the immediate precursor of histamine). These “upstream” enzymes in the pathway include: an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase, including any isoforms, paralogs, or orthologs having these enzymatic activities (which as those of skill in the art readily appreciate may be known by different names). The at least one additional alteration can increase the activity of the upstream pathway enzyme(s) by any available means, e.g., by: (1) modulating the expression or activity of the native enzyme(s), (2) expressing one or more additional copies of the genes for the native enzymes, and/or (3) expressing one or more copies of the genes for one or more non-native enzymes.

In some embodiments, increased flux through the pathway can be achieved by expressing one or more genes encoding a feedback-deregulated enzyme, as discussed above. For example, the engineered host cell can include and express one or more feedback-deregulated ATP phosphoribosyltransferase genes.

The engineered microbial cells can contain introduced genes that have a native nucleotide sequence or that differ from native. For example, the native nucleotide sequence can be codon-optimized for expression in a particular host cell. The amino acid sequences encoded by any of these introduced genes can be native or can differ from native. In various embodiments, the amino acid sequences have at least 60 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a native amino acid sequence.

In some embodiments, increased availability of precursors to histamine can be achieved by reducing the expression or activity of enzymes that consume one or more histamine pathway precursors, such as an enolase, a pyruvate dehydrogenase, a pentose phosphate pathway sugar isomerase, a transaldolase, a transketolase, a ribulose-5-phosphate epimerase, and a aribulose-5-phosphate isomerase. For example, the engineered host cell can include one or more promoter swaps to down-regulate expression of any of these enzymes and/or can have their genes deleted to eliminate their expression entirely.

The approach described herein has been carried out in bacterial cells, namely C. glutamicum and B. subtilis (prokaryotes) and in fungal cells, namely the yeasts S. cerevisiae and Y. lypolytica (eukaryotes). (See Examples 1 and 2.)

Illustrative Engineered Yeast Cells

In certain embodiments, the engineered yeast (e.g., S. cerevisiae) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Chromobacterium sp. LK1 (e.g., SEQ ID NO:6). In particular embodiments, the Chromobacterium sp. LK1 histamine decarboxylase can include SEQ ID NO:6. The engineered yeast (e.g., S. cerevisiae) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae (SEQ ID NO:3). In particular embodiments, the S. cerevisiae ATP phosphoribosyltransferase includes SEQ ID NO:3.

In certain embodiments, the engineered yeast (e.g., Y. lipolytica) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Acinetobacter baumannii strain AB0057 (e.g., SEQ ID NO:1). In particular embodiments, the Acinetobacter baumannii strain AB0057 histamine decarboxylase can include SEQ ID NO:1. The engineered yeast (e.g., Y. lipolytica) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3). In particular embodiments, the S. cerevisiae S288c ATP phosphoribosyltransferase includes SEQ ID NO:3.

These may be the only genetic alterations of the engineered yeast cell, or the yeast cell can include one or more additional genetic alterations, as discussed more generally above.

For example, in particular embodiments, the engineered yeast S. cerevisiae cell described above additionally expresses a feedback deregulated variant of a C. glutamicum ATP phosphoribosyltransferase, which typically has at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, or 95 percent amino acid sequence identity to a variant of a C. glutamicum ATP phosphoribosyltransferase containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO:7) In particular embodiments, the C. glutamicum ATP phosphoribosyltransferase variant can include SEQ ID NO:7.

Illustrative Engineered Bacterial Cells

In certain embodiments, the engineered bacterial (e.g., C. glutamicum) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Acinetobacter baumannii (e.g., SEQ ID NO:1). In particular embodiments, the Acinetobacter baumannii histamine decarboxylase can include SEQ ID NO:1. The engineered bacterial (e.g., C. glutamicum) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c (SEQ ID NO:3). In particular embodiments, the S. cerevisiae S288c ATP phosphoribosyltransferase includes SEQ ID NO:3. In some embodiments, the engineered bacterial (e.g., C. glutamicum) cell expresses, instead of the ATP phosphoribosyltransferase, an imidazole-glycerol phosphate dehydratase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to an imidazole-glycerol phosphate dehydratase from C. glutamicum (SEQ ID NO:2). In particular embodiments, the C. glutamicum imidazole-glycerol phosphate dehydratase includes SEQ ID NO:2.

In certain embodiments, the engineered bacterial (e.g., B. subtilis) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Lactobacillus sp. (strain 30a) (e.g., SEQ ID NO:4). In particular embodiments, the Lactobacillus sp. (strain 30a) histamine decarboxylase can include SEQ ID NO:4. The engineered bacterial (e.g., B. subtilis) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (SEQ ID NO:5). In particular embodiments, the Salmonella typhimurium LT2 ATP phosphoribosyltransferase includes SEQ ID NO:5.

Culturing of Engineered Microbial Cells

Any of the microbial cells described herein can be cultured, e.g., for maintenance, growth, and/or histamine production.

In some embodiments, the cultures are grown to an optical density at 600 nm of 10-500, such as an optical density of 50-150.

In various embodiments, the cultures include produced histamine at titers of at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L, or at least 1, 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 g/L. In various embodiments, the titer is in the range of 10 μg/L to 10 g/L, 25 μg/L to 20 g/L, 100 μg/L to 10 g/L, 200 μg/L to 5 g/L, 500 μg/L to 4 g/L, 1 mg/L to 3 g/L, 500 mg/L to 2 g/L or any range bounded by any of the values listed above.

Culture Media

Microbial cells can be cultured in any suitable medium including, but not limited to, a minimal medium, i.e., one containing the minimum nutrients possible for cell growth. Minimal medium typically contains: (1) a carbon source for microbial growth; (2) salts, which may depend on the particular microbial cell and growing conditions; and (3) water. Suitable media can also include any combination of the following: a nitrogen source for growth and product formation, a sulfur source for growth, a phosphate source for growth, metal salts for growth, vitamins for growth, and other cofactors for growth.

Any suitable carbon source can be used to cultivate the host cells. The term “carbon source” refers to one or more carbon-containing compounds capable of being metabolized by a microbial cell. In various embodiments, the carbon source is a carbohydrate (such as a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide), or an invert sugar (e.g., enzymatically treated sucrose syrup). Illustrative monosaccharides include glucose (dextrose), fructose (levulose), and galactose; illustrative oligosaccharides include dextran or glucan, and illustrative polysaccharides include starch and cellulose. Suitable sugars include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). Other, less expensive carbon sources include sugar cane juice, beet juice, sorghum juice, and the like, any of which may, but need not be, fully or partially deionized.

The salts in a culture medium generally provide essential elements, such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize proteins and nucleic acids.

Minimal medium can be supplemented with one or more selective agents, such as antibiotics.

To produce histamine, the culture medium can include, and/or is supplemented during culture with, glucose and/or a nitrogen source such as urea, an ammonium salt, ammonia, or any combination thereof.

Culture Conditions

Materials and methods suitable for the maintenance and growth of microbial cells are well known in the art. See, for example, U.S. Pub. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2004/033646, WO 2009/076676, WO 2009/132220, and WO 2010/003007, Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass.

In general, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as about 20° C. to about 37° C., about 6% to about 84% CO₂, and a pH between about 5 to about 9). In some aspects, cells are grown at 35° C. In certain embodiments, such as where thermophilic bacteria are used as the host cells, higher temperatures (e.g., 50° C.-75° C.) may be used. In some aspects, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Cells can be grown under aerobic, anoxic, or anaerobic conditions based on the requirements of the particular cell.

Standard culture conditions and modes of fermentation, such as batch, fed-batch, or continuous fermentation that can be used are described in U.S. Publ. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2009/076676, WO 2009/132220, and WO 2010/003007. Batch and Fed-Batch fermentations are common and well known in the art, and examples can be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.

In some embodiments, the cells are cultured under limited sugar (e.g., glucose) conditions. In various embodiments, the amount of sugar that is added is less than or about 105% (such as about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of the amount of sugar that can be consumed by the cells. In particular embodiments, the amount of sugar that is added to the culture medium is approximately the same as the amount of sugar that is consumed by the cells during a specific period of time. In some embodiments, the rate of cell growth is controlled by limiting the amount of added sugar such that the cells grow at the rate that can be supported by the amount of sugar in the cell medium. In some embodiments, sugar does not accumulate during the time the cells are cultured. In various embodiments, the cells are cultured under limited sugar conditions for times greater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours or even up to about 5-10 days. In various embodiments, the cells are cultured under limited sugar conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time the cells are cultured. While not intending to be bound by any particular theory, it is believed that limited sugar conditions can allow more favorable regulation of the cells.

In some aspects, the cells are grown in batch culture. The cells can also be grown in fed-batch culture or in continuous culture. Additionally, the cells can be cultured in minimal medium, including, but not limited to, any of the minimal media described above. The minimal medium can be further supplemented with 1.0% (w/v) glucose (or any other six-carbon sugar) or less. Specifically, the minimal medium can be supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose. In some cultures, significantly higher levels of sugar (e.g., glucose) are used, e.g., at least 10% (w/v), 20% (w/v), 30% (w/v), 40% (w/v), 50% (w/v), 60% (w/v), 70% (w/v), or up to the solubility limit for the sugar in the medium. In some embodiments, the sugar levels falls within a range of any two of the above values, e.g.: 0.1-10% (w/v), 1.0-20% (w/v), 10-70% (w/v), 20-60% (w/v), or 30-50% (w/v). Furthermore, different sugar levels can be used for different phases of culturing. For fed-batch culture (e.g., of S. cerevisiae or C. glutamicum), the sugar level can be about 100-200 g/L (10-20% (w/v)) in the batch phase and then up to about 500-700 g/L (50-70% in the feed).

Additionally, the minimal medium can be supplemented 0.1% (w/v) or less yeast extract. Specifically, the minimal medium can be supplemented with 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. Alternatively, the minimal medium can be supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), or 0.02% (w/v) yeast extract. In some cultures, significantly higher levels of yeast extract can be used, e.g., at least 1.5% (w/v), 2.0% (w/v), 2.5% (w/v), or 3% (w/v). In some cultures (e.g., of S. cerevisiae or C. glutamicum), the yeast extract level falls within a range of any two of the above values, e.g.: 0.5-3.0% (w/v), 1.0-2.5% (w/v), or 1.5-2.0% (w/v).

Illustrative materials and methods suitable for the maintenance and growth of the engineered microbial cells described herein can be found below in Example 1.

Histamine Production and Recovery

Any of the methods described herein may further include a step of recovering histamine. In some embodiments, the produced histamine contained in a so-called harvest stream is recovered/harvested from the production vessel. The harvest stream may include, for instance, cell-free or cell-containing aqueous solution coming from the production vessel, which contains histamine as a result of the conversion of production substrate by the resting cells in the production vessel. Cells still present in the harvest stream may be separated from the histamine by any operations known in the art, such as for instance filtration, centrifugation, decantation, membrane crossflow ultrafiltration or microfiltration, tangential flow ultrafiltration or microfiltration or dead end filtration. After this cell separation operation, the harvest stream is essentially free of cells.

Further steps of separation and/or purification of the produced histamine from other components contained in the harvest stream, i.e., so-called downstream processing steps may optionally be carried out. These steps may include any means known to a skilled person, such as, for instance, concentration, extraction, crystallization, precipitation, adsorption, ion exchange, and/or chromatography. Any of these procedures can be used alone or in combination to purify histamine. Further purification steps can include one or more of, e.g., concentration, crystallization, precipitation, washing and drying, treatment with activated carbon, ion exchange, nanofiltration, and/or re-crystallization. The design of a suitable purification protocol may depend on the cells, the culture medium, the size of the culture, the production vessel, etc. and is within the level of skill in the art.

The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. Changes therein and other uses which are encompassed within the spirit of the disclosure, as defined by the scope of the claims, will be identifiable to those skilled in the art.

Example 1—Construction and Selection of Strains of Corynebacteria glutamicum and Saccharomyces cerevisiae Engineered to Produce Histamine

Plasmid/DNA Design

All strains tested for this work were transformed with plasmid DNA designed using proprietary software. Plasmid designs were specific to each of the host organisms engineered in this work. The plasmid DNA was physically constructed by a standard DNA assembly method. This plasmid DNA was then used to integrate metabolic pathway inserts by one of two host-specific methods, each described below.

C. glutamicum Pathway Integration

A “loop-in, single-crossover” genomic integration strategy has been developed to engineer C. glutamicum strains. FIG. 14 illustrates genomic integration of loop-in only and loop-in/loop-out constructs and verification of correct integration via colony PCR. Loop-in only constructs (shown under the heading “Loop-in”) contained a single 2-kb homology arm (denoted as “integration locus”), a positive selection marker (denoted as “Marker”)), and gene(s) of interest (denoted as “promoter-gene-terminator”). A single crossover event integrated the plasmid into the C. glutamicum chromosome. Integration events are stably maintained in the genome by growth in the presence of antibiotic (25p g/ml kanamycin). Correct genomic integration in colonies derived from loop-in integration were confirmed by colony PCR with UF/IR and DR/IF PCR primers.

Loop-in, loop-out constructs (shown under the heading “Loop-in, loop-out) contained two 2-kb homology arms (5′ and 3′ arms), gene(s) of interest (arrows), a positive selection marker (denoted “Marker”), and a counter-selection marker. Similar to “loop-in” only constructs, a single crossover event integrated the plasmid into the chromosome of C. glutamicum. Note: only one of two possible integrations is shown here. Correct genomic integration was confirmed by colony PCR and counter-selection was applied so that the plasmid backbone and counter-selection marker could be excised. This results in one of two possibilities: reversion to wild-type (lower left box) or the desired pathway integration (lower right box). Again, correct genomic loop-out is confirmed by colony PCR. (Abbreviations: Primers: UF=upstream forward, DR=downstream reverse, IR=internal reverse, IF=internal forward.)

S. cerevisiae Pathway Integration

A “split-marker, double-crossover” genomic integration strategy has been developed to engineer S. cerevisiae strains. FIG. 11 illustrates genomic integration of complementary, split-marker plasmids and verification of correct genomic integration via colony PCR in S. cerevisiae. Two plasmids with complementary 5′ and 3′ homology arms and overlapping halves of a URA3 selectable marker (direct repeats shown by the hashed bars) were digested with meganucleases and transformed as linear fragments. A triple-crossover event integrated the desired heterologous genes into the targeted locus and re-constituted the full URA3 gene. Colonies derived from this integration event were assayed using two 3-primer reactions to confirm both the 5′ and 3′ junctions (UF/IF/wt-R and DR/IF/wt-F). For strains in which further engineering is desired, the strains can be plated on 5-FOA plates to select for the removal of URA3, leaving behind a small single copy of the original direct repeat. This genomic integration strategy can be used for gene knock-out, gene knock-in, and promoter titration in the same workflow.

Cell Culture

The workflow established for S. cerevisiae involved a hit-picking step that consolidated successfully built strains using an automated workflow that randomized strains across the plate. For each strain that was successfully built, up to four replicates were tested from distinct colonies to test colony-to-colony variation and other process variation. If fewer than four colonies were obtained, the existing colonies were replicated so that at least four wells were tested from each desired genotype.

The colonies were consolidated into 96-well plates with selective medium (SD-ura for S. cerevisiae) and cultivated for two days until saturation and then frozen with 16.6% glycerol at −80° C. for storage. The frozen glycerol stocks were then used to inoculate a seed stage in minimal media with a low level of amino acids to help with growth and recovery from freezing. The seed plates were grown at 30° C. for 1-2 days. The seed plates were then used to inoculate a main cultivation plate with minimal medium and grown for 48-88 hours. Plates were removed at the desired time points and tested for cell density (OD600), viability and glucose, supernatant samples stored for LC-MS analysis for product of interest.

Cell Density

Cell density was measured using a spectrophotometric assay detecting absorbance of each well at 600 nm. Robotics were used to transfer fixed amounts of culture from each cultivation plate into an assay plate, followed by mixing with 175 mM sodium phosphate (pH 7.0) to generate a 10-fold dilution. The assay plates were measured using a Tecan M1000 spectrophotometer and assay data uploaded to a LIMS database. A non-inoculated control was used to subtract background absorbance. Cell growth was monitored by inoculating multiple plates at each stage, and then sacrificing an entire plate at each time point.

To minimize settling of cells while handling large number of plates (which could result in a non-representative sample during measurement) each plate was shaken for 10-15 seconds before each read. Wide variations in cell density within a plate may also lead to absorbance measurements outside of the linear range of detection, resulting in underestimate of higher OD cultures. In general, the tested strains so far have not varied significantly enough for this be a concern.

Liquid-Solid Separation

To harvest extracellular samples for analysis by LC-MS, liquid and solid phases were separated via centrifugation. Cultivation plates were centrifuged at 2000 rpm for 4 minutes, and the supernatant was transferred to destination plates using robotics. 75p L of supernatant was transferred to each plate, with one stored at 4° C., and the second stored at 80° C. for long-term storage.

First-Round Genetic Engineering Results in Corynebacteria glutamicum and Saccharomyces cerevisiae

A library approach was taken to screen heterologous pathway enzymes to establish the histamine pathway. For histidine decarboxylase, 18 heterologous sequences were tested from Bacteria, Archaea, Viridiplantae, Vertebrata, Metazoa, and Arthropoda sources listed in Table 1. The histidine decarboxylases were codon-optimized and expressed in both Saccharomyces cerevisiae and Corynebacteria glutamicum hosts.

Histidine biosynthesis is subject to feedback inhibition, therefore a feedback deregulated ATP phosphoribosyltransferase was tested with the histidine decarboxylases to improve production of histidine, the substrate for histidine decarboxylase. The ATP phosphoribosyltransferases tested were from Salmonella typhimurium and Corynebacteria glutamicum, harboring known deletions and point mutations that render them resistant to feedback inhibition.

First-round genetic engineering results are shown in Table 1 and FIGS. 2 (C. glutamicum) and 3 (S. cerevisiae).

TABLE 1 First-round genetic engineering results in Corynebacteria glutamicum and Saccharomyces cerevisiae E1 E2 Enzyme Enzyme Codon Enzyme Enzyme Codon E1 1— 1— Optimi- E2 2— E2 2— Optimi- Strain Titer Uniprot activity source zation Uniprot activity Modifi- source zation name (μg/L) ID name organism Abbrev. ID name cations organism Abbrev. Corynebacterium glutamicum CgHISM 985.0 E3QMN8 histidine Methanosarcina Cg N_06 decarboxylase barkeri str. Wiesmoor CgHISM 695.7 Q467R8 histidine Methanosarcina Cg N_07 decarboxylase barkeri (strain Fusaro / DSM 804) CgHISM 385.0 P00862 histidine Lactobacillus sp. Cg N_08 decarboxylase (strain 30a) CgHISM 14370.2 B71459 histidine Acinetobacter Cg N_11 decarboxylase baumannii (strain AB0057) CgHISM 401.1 E3QMN8 histidine Methanosarcina Cg N_13 decarboxylase barkeri str. Wiesmoor CgHISM 7529.8 P00862 histidine Lactobacillus Cg P00499 ATP Deletion of Salmonella Cg N_15 decarboxylase sp. phosphoribosyl- Q207-E208 typhimurium (strain 30a) transferase CgHISM 4.0 P00862 histidine Lactobacillus Cg P00499 ATP Deletion of Salmonella Cg N_16 decarboxylase sp. phosphoribosyl- Q207-E208 typhimurium (strain 30a) transferase CgHISM 3.9 P23738 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Cg N_17 decarboxylase barkeri (strain phosphoribosyl- Q207-E208 typhimurium Fusaro / transferase DSM 804) CgHISM 75.0 Q05733 histidine Drosophila Cg P00499 ATP Deletion of Salmonella Cg N_19 decarboxylase melanogaster phosphoribosyl- Q207-E208 typhimurium transferase CgHISM 3.8 J6KM89 histidine Chromo- Cg P00499 ATP Deletion of Salmonella Cg N_24 decarboxylase bacterium phosphoribosyl- Q207-E208 typhimurium sp. LK1 transferase CgHISM 1.7 E3QMN8 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Cg N_25 decarboxylase barkeri str. phosphoribosyl- Q207-E208 typhimurium Wiesmoor transferase CgHISM 458.5 E3QMN8 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Cg N_26 decarboxylase barkeri str. phosphoribosyl- Q207-E208 typhimurium Wiesmoor transferase CgHISM 462.1 Q467R8 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Cg N_27 decarboxylase barkeri (strain phosphoribosyl- Q207-E208 typhimurium Fusaro / transferase DSM 804) CgHISM 1258.2 Q467R8 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Cg N_28 decarboxylase barkeri (strain phosphoribosyl- Q207-E208 typhimurium Fusaro / transferase DSM 804) CgHISM 2126.4 P00862 histidine Lactobacillus sp. Cg P00499 ATP Deletion of Salmonella Cg N_30 decarboxylase (strain 30a) phosphoribosyl- Q207-E208 typhimurium transferase CgHISM 234.7 Q05733 histidine Drosophila Cg P00499 ATP Deletion of Salmonella Cg N_33 decarboxylase melanogaster phosphoribosyl- Q207-E208 typhimurium transferase CgHISM 11905.3 B71459 histidine Acinetobacter Cg P00499 ATP Deletion of Salmonella Cg N_35 decarboxylase baumannii (strain phosphoribosyl- Q207-E208 typhimurium AB0057) transferase CgHISM 615.0 E3QMN8 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Cg N_39 decarboxylase barkeri str. phosphoribosyl- Q207-E208 typhimurium Wiesmoor transferase Saccharomyces cerevisiae ScHISM 36145.0 P00862 histidine Lactobacillus Sc Q9Z472 ATP N215K, Coryne- Sc N_16 decarboxylase sp. phosphoribosyl- L231F, bacterium (strain 30a) transferase T235A glutamicum ScHISM 2369.9 P54772 histidine Solanum Sc P00499 ATP Deletion of Salmonella Sc N_17 decarboxylase lycopersicum phosphoribosyl- Q207-E208 typhimurium transferase ScHISM 1747.7 P23738 histidine Mus musculus Sc P00499 ATP Deletion of Salmonella Cg N_18 decarboxylase phosphoribosyl- Q207-E208 typhimurium transferase ScHISM 2432.4 Q05733 histidine Drosophila Sc P00499 ATP Deletion of Salmonella Sc N_20 decarboxylase melanogaster phosphoribosyl- Q207-E208 typhimurium transferase ScHISM 43606.9 J6KM89 histidine Chromo- Sc P00499 ATP Deletion of Salmonella Cg N_21 decarboxylase bacterium phosphoribosyl- Q207-E208 typhimurium sp. LK1 transferase ScHISM 43021.9 E3QMN8 histidine Methanosarcina Sc Q9Z472 ATP N215K, Coryne- Sc N_22 decarboxylase barkeri str. phosphoribosyl- L231F, bacterium Wiesmoor transferase T235A glutamicum ScHISM 36145.8 Q467R8 histidine Methanosarcina Sc P00499 ATP Deletion of Salmonella Sc N_23 decarboxylase barkeri (strain phosphoribosyl- Q207-E208 typhimurium Fusaro / transferase DSM 804) ScHISM 47208.0 P00862 histidine Lactobacillus Cg P00499 ATP Deletion of Salmonella Cg N_24 decarboxylase sp. phosphoribosyl- Q207-E208 typhimurium (strain 30a) transferase ScHISM 3130.1 P23738 histidine Mus musculus Cg Q9Z472 ATP N215K, Coryne- Sc N_25 decarboxylase phosphoribosyl- L231F, bacterium transferase T235A glutamicum ScHISM 3262.5 Q05733 histidine Drosophila Cg P00499 ATP Deletion of Salmonella Sc N_26 decarboxylase melanogaster phosphoribosyl- Q207-E208 typhimurium transferase ScHISM 90811.0 J6KM89 histidine Chromo- Cg Q9Z472 ATP N215K, Coryne- Sc N_28 decarboxylase bacterium phosphoribosyl- L231F, T235A bacterium sp. LK1 transferase glutamicum ScHISM 42708.8 E3QMN8 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Sc N_29 decarboxylase barkeri str. phosphoribosyl- Q207-E208 typhimurium Wiesmoor transferase ScHISM 27660.1 Q467R8 histidine Methanosarcina Cg P00499 ATP Deletion of Salmonella Cg N_30 decarboxylase barkeri phosphoribosyl- Q207-E208 typhimurium (strain transferase Fusaro / DSM 804) ScHISM 33356.6 P00862 histidine Lactobacillus Sc Q9Z472 ATP N215K, Coryne- Sc N_31 decarboxylase sp. phosphoribosyl- L231F, bacterium (strain 30a) transferase T235A glutamicum ScHISM 711.5 P54772 histidine Solanum Sc P00499 ATP Deletion of Salmonella Sc N_32 decarboxylase lycopersicum phosphoribosyl- Q207-E208 typhimurium transferase ScHISM 1523.1 P23738 histidine Mus musculus Sc P00499 ATP Deletion of Salmonella Cg N_33 decarboxylase phosphoribosyl- Q207-E208 typhimurium transferase ScHISM 43170.7 E3QMN8 histidine Methanosarcina Sc Q9Z472 ATP N215K, Coryne- Sc N_37 decarboxylase barkeri str. phosphoribosyl- L231F, bacterium Wiesmoor transferase T235A glutamicum ScHISM 30675.5 Q467R8 histidine Methanosarcina Sc P00499 ATP Deletion of Salmonella Sc N_38 decarboxylase barkeri (strain phosphoribosyl- Q207-E208 typhimurium Fusaro / transferase DSM 804) ScHISM 38293.2 P00862 histidine Lactobacillus Cg P00499 ATP Deletion of Salmonella Cg N_39 decarboxylase sp. phosphoribosyl- Q207-E208 typhimurium (strain 30a) transferase Note: “Cg” refers to codon optimization for Corynebacterium glutamicum; “Sc” refers to codon optimization for Saccharomyces cerevisiae.

Second-Round Genetic Engineering Results in Corynebacteria glutamicum and Saccharomyces cerevisiae

A library approach was taken to improve histamine production by separately expressing each upstream pathway enzyme with a constitutive promoter to screen for the rate-limiting step. The histidine pathway enzymes screened are listed in Table 2. In addition, the enzymes in Table 2, the strains contained the best enzymes from first round: the Corynebacteria glutamicum host contains histidine decarboxylase (UniProt ID B7I459) (SEQ ID NO: 1) and ATP phosphoribosyltransferase (UniProt ID P00499) (SEQ ID NO: 5) containing the deletion Q207-E208, and the Saccharomyces cerevisiae host contains histidine decarboxylase (UniProt ID J6KM89)(SEQ ID NO: 6) and ATP phosphoribosyltransferase (UniProt ID Q9Z472) (SEQ ID NO: 7) containing the amino acid substitutions N215K, L231F and T235A.

Second-round genetic engineering results are shown in Table 2 and FIGS. 4 (C. glutamicum) and 5 (S. cerevisiae).

In C. glutamicum, a titer of 24 mg/L was achieved after two rounds of engineering from the integration of two genes: a histidine decarboxylase gene from Acinetobacter baumannii, and constitutive expression of an imidazoleglycerol-phosphate dehydratase from C. glutamicum.

In S. cerevisiae, a titer of 385 mg/L was achieved in two rounds of engineering from the integration of three genes: a histidine decarboxylase gene from Chromobacterium sp. LK1 (SEQ ID NO: 6), an ATP phosphoribosyltransferase from C. glutamicum containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO: 7), and a constitutively expressed ATP phosphoribosyltransferase from S. cerevisiae (SEQ ID NO: 3).

TABLE 2 Second-round genetic engineering results in genetic engineering results in Corynebacteria glutamicum and Saccharomyces cerevisiae E1 Codon Titer E1 Enzyme 1- Optimization Strain name (μg/L) Uniprot ID Enzyme 1-activity name source organism Abbrev. Corynebacteria glutamicum CgHISMN_41 13702.1 O68602 1-(5-phosphoribosyl)5[(5- Corynebacterium Native phosphoribosylamino) glutamicum methylideneamino] imidazole-4- carboxamide isomerase CgHISMN_42 12671.2 Q9KJU3 Imidazoleglycerol- Corynebacterium Native phosphate dehydratase glutamicum CgHISMN_43 11800.4 Q9KJU4 Histidinol-phosphate Corynebacterium Native aminotransferase glutamicum CgHISMN_44 8667.2 Q8NNT5 Histidinol dehydrogenase Corynebacterium Native glutamicum CgHISMN_45 12375.3 Q9Z471 Phosphoribosyl- Corynebacterium Native ATP pyrophosphatase glutamicum CgHISMN_46 10963.6 O31139 Imidazole glycerol Corynebacterium Native phosphate synthase subunit glutamicum CgHISMN_47 16246.0 O69043 Imidazole glycerol Corynebacterium Native phosphate synthase subunit glutamicum CgHISMN_48 13038.8 Q9Z472 ATP phosphoribosyltransferase Corynebacterium Native glutamicum CgHISMN_49 10749.0 Q8NNT9 phosphoribosyl- Corynebacterium Native AMP cyclohydrolase glutamicum CgHISMN_50 12960.8 O68602 1-(5-phosphoribosyl)5[(5- Corynebacterium Native phosphoribosylamino) glutamicum methylideneamino] imidazole-4- carboxamide isomerase CgHISMN_51 9958.4 Q9KJU3 Imidazoleglycerol- Corynebacterium Native phosphate dehydratase glutamicum CgHISMN_52 18963.0 Q9KJU4 Histidinol-phosphate Corynebacterium Native aminotransferase glutamicum CgHISMN_53 20328.9 Q8NNT5 Histidinol dehydrogenase Corynebacterium Native glutamicum CgHISMN_54 20051.4 O31139 Imidazole glycerol phosphate Corynebacterium Native synthase subunit glutamicum CgHISMN_55 15070.9 O69043 Imidazole glycerol phosphate Corynebacterium Native synthase subunit glutamicum CgHISMN_56 12799.1 O68602 1-(5-phosphoribosyl)5[(5- Corynebacterium Native phosphoribosylamino) glutamicum methylideneamino] imidazole-4- carboxamide isomerase CgHISMN_57 24773.6 Q9KJU3 Imidazoleglycerol- Corynebacterium Native phosphate dehydratase glutamicum CgHISMN_58 15268.6 Q9KJU4 Histidinol-phosphate Corynebacterium Native aminotransferase glutamicum CgHISMN_59 12555.0 Q8NNT5 Histidinol dehydrogenase Corynebacterium Native glutamicum CgHISMN_60 17725.6 Q9Z471 Phosphoribosyl- Corynebacterium Native ATP pyrophosphatase glutamicum CgHISMN_61 18777.4 O69043 Imidazole glycerol Corynebacterium Native phosphate synthase subunit glutamicum CgHISMN_62 19782.8 Q9Z472 ATP phosphoribosyltransferase Corynebacterium Native glutamicum CgHISMN_63 15092.7 Q8NNT9 phosphoribosyl- Corynebacterium Native AMP cyclohydrolase glutamicum Saccharomyces cerevisiae ScHISMN_41 385518.2 P00498 ATP phosphoribosyltransferase Saccharomyces Native cerevisiae ScHISMN_42 70003.1 P00815 histidinol dehydrogenase, Saccharomyces Native phosphoribosyl-AMP cyclohydrolase, cerevisiae phosphoribosyl-ATP diphosphatase ScHISMN_43 75039.5 P33734 Imidazole glycerol phosphate Saccharomyces Native synthase subunit HisF cerevisiae ScHISMN_44 71402.5 P07172 histidinol-phosphate transaminase Saccharomyces Native cerevisiae ScHISMN_46 64866.5 P06633 Imidazoleglycerol- Saccharomyces Native phosphate dehydratase cerevisiae ScHISMN_48 113026.6 P00498 ATP phosphoribosyltransferase Saccharomyces Native cerevisiae ScHISMN_49 79488.5 P00815 histidinol dehydrogenase, Saccharomyces Native phosphoribosyl-AMP cyclohydrolase, cerevisiae phosphoribosyl-ATP diphosphatase ScHISMN_50 92719.6 P33734 Imidazole glycerol phosphate Saccharomyces Native synthase subunit HisF cerevisiae ScHISMN_51 88847.1 P07172 histidinol-phosphate transaminase Saccharomyces Native cerevisiae ScHISMN_52 70650.9 P38635 histidinol-phosphatase Saccharomyces Native cerevisiae ScHISMN_53 74127.8 P06633 Imidazoleglycerol- Saccharomyces Native phosphate dehydratase cerevisiae ScHISMN_56 73080.2 P00815 histidinol dehydrogenase, Saccharomyces Native phosphoribosyl-AMP cyclohydrolase, cerevisiae phosphoribosyl-ATP diphosphatase ScHISMN_57 78656.1 P33734 Imidazole glycerol phosphate Saccharomyces Native synthase subunit HisF cerevisiae ScHISMN_58 69769.0 P07172 histidinol-phosphate transaminase Saccharomyces Native cerevisiae ScHISMN_59 59139.1 P38635 histidinol-phosphatase Saccharomyces Native cerevisiae ScHISMN_60 65506.7 P06633 Imidazoleglycerol- Saccharomyces Native phosphate dehydratase cerevisiae

Third-Round Genetic Engineering Results in Saccharomyces cerevisiae

Histamine production was further pursued in S. cerevisiae, and we designed plasmids to integrate additional copies of upstream pathway genes expressed by a strong constitutive promoter to avoid native regulation of a gene (Table 3). An expanded search was undertaken to test additional histidine decarboxylases that have similar sequences to the enzymes initially identified as active (Table 3).

In parallel we pursued modulating native gene expression to further improve histamine production. Our engineering approach was to take a best S. cerevisiae strain from the second round and test either a strong or weak constitutive promoter in place of the native promoter. Gene targets for promoter changes were selected to redirect flux supply precursors to histidine. Strain designs being tested include designs for increasing pentose phosphate pathway flux by expressing a non-native feedback deregulated glucose-6-phosphate dehydrogenase (zwf) and decreasing the “lower” pentose phosphate pathway flux thru the sugar isomerase enzymes.

Promoter replacements for lower expression of genes that are thought to be essential (i.e., cannot be deleted), but were expected to increase the upper glycolysis metabolite pool available for histamine production, targeted: 1) enolase (Eno2), to reduce flux through lower glycolysis, 2) pyruvate dehydrogenase (PDH, Lpd1) for lower flux through the C3/C2 node, and 3) pentose phosphate pathway sugar isomerases, which use the histamine metabolite precursor ribose-5-phosphate (Tall). An illustrative list of promoter-swap (“proswap”) and deletion (“knockout”) targets in S. cerevisiae includes:

Annotation_name Type Promoter_name Gene_name YDR380W knockout Aro10 YDL047W knockout Sit4 YML035C knockout Amd1 YMR020W knockout Fms1 YNL229C knockout Ure2 YJL052W proswap pRnr1 Tdh1 YJR009C proswap pRnr1 Tdh2 YGR192C proswap pRnr1 Tdh3 YFL018C proswap pRnr1 Lpd1 YHR174W proswap pRnr1 Eno2 YNR001C proswap pRnr1 Cit1 YCR012W proswap pRnr1 Pgk1 YLR354C proswap pRnr1 Tal1 YBR117C proswap pRnr1 Tkl2 YPR074C proswap pRnr1 Tkl1 YML035C proswap pRev1 Amd1 YHR216W proswap pRev1 Imd2 YOR155C proswap pRev1 Isn1 YNL229C proswap pRev1 Ure2 YER086W proswap pRnr1 Ilv1 YDR380W proswap pRnr1 Aro10 YEL009C proswap pRev1 Gcn4

Promoters were selected based on expression data from Lee et al [7].

Additional genetic engineering results for S. cerevisiae are shown in Table 3 and FIG. 10. The parent strain for the strain designs shown in Table 3 (also the reference strain, ScHISMN_41) contained a histidine decarboxylase (UniProt ID J6KM89) and an ATP phosphoribosyltransferase (UniProt ID Q9Z472) harboring the amino acid substitutions N215K, L231F and T235A, and the ATP phosphoribosyltransferase from S. cerevisiae. The reference strain had a histamine titer of 131 mg/L.

Improved titer was observed in strains that expressed each of the following enzymes from a strong constitutive promoter:

1. Transketolase (EC 2.2.1.1) (SEQ ID NO: 27), which catalyzes the interconversion of sugars in the pentose phosphate pathway and produces ribose-5-phosphate, which is a precursor to PPRP, the initial metabolite in the histidine biosynthesis pathway.

2. Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) (SEQ ID NO: 28) (highest titer: 191 mg/L relative to control in experiment 131 mg/L).

3. ATP phosphoribosyltransferase (EC 2.4.2.17) (SEQ ID NO: 3).

4. Trifunctional histidinol dehydrogenase (EC 1.1.1.23)/phosphoribosyl-AMP cyclohydrolase (EC 3.5.4.19)/phosphoribosyl-ATP diphosphatase (EC 3.6.1.31) (SEQ ID NO: 29).

5. Histidinol-phosphate aminotransferase (EC 2.6.1.9) (SEQ ID NO: 14).

6. 5′ProFAR isomerase (EC 5.3.1.16) (SEQ ID NO: 31).

7. Imidazole glycerol phosphate synthase (EC 4.3.1.B2) (SEQ ID NO: 21).

8. Triose-phosphate isomerase (EC 5.3.1.1), harboring the amino acid substitutions harboring the amino acid substitutions I170V (SEQ ID NO: 32) or I170T [8].

9. Glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49), harboring the amino acid substitution A243T (SEQ ID NO: 26).

10. Various histidine decarboxylases (EC 4.1.1.22):

-   -   a. UniProt ID A0A089YPE5 (SEQ ID NO: 33)     -   b. UniProt ID A0A126S6G9 (SEQ ID NO: 34)     -   c. UniProt ID A0A0A1R6V3 (SEQ ID NO: 35)     -   d. UniProt ID A0A1W0CM88 (SEQ ID NO: 36)     -   e. UniProt ID P00862 (SEQ ID NO: 4)     -   f. UniProt ID A0A0K6GJ74 (SEQ ID NO: 37)     -   g. UniProt ID T0QL99 (SEQ ID NO: 38)     -   h. UniProt ID A0A1B8HLR1 (SEQ ID NO: 39)

TABLE 3 Third-round genetic engineering results in Saccharomyces cerevisiae Built and tested strain designs: Enzyme E1 Enzyme Enzyme Enzyme Enzyme E3 Enzyme E1 1— Modi- 1— E2 2— 2— E3 3— Modi- 3— Strain Titer Uniprot activity fica- source Uniprot activity source Uniprot activity fica- source name (μg/L) ID name tions organism ID name organism ID name tions organism Sc 39059 A0A0C Histidine Lactobacillus HISM 1PR48 decarboxy- fructivorans N_100 lase ScHIS 144871 T0QL99 Histidine Aeromonas MN_ decarboxy- salmonicida 101 lase subsp. pectinolytica 34mel ScHIS 143763 A0A1B8 Histidine Morganella MN_ HLR1 decarboxy- psychrotoler- 102 lase ans ScHIS 155931 Q9Z472 ATP Coryne- P00815 tri- Saccharo- P0A717 Ribose- Escheri- MN_ phosphori- bacterium functional myces phos- chia l03 bosyl- glutamicum histidinol cerevisiae phate coli transferase (strain dehydro- S288c pyro- (strain ATCC genase/ phos- K12) 13032 / phos- pho- DSM phoribo- kinase 20300 / syl-AMP JCM cyclo- 1318 / hydrolase/ LMG phos- 3730 / phoribo- NCIMB syl-ATP 10025) diphos- phatase ScHIS 151846 P0A717 Ribose- Escherichia MN_ phosphate coli 104 pyrophos- (strain pho- K12) kinase ScHIS 191110 Q12265 Ribose- Saccharo- P38620 Ribose- Saccharo- Q680A5 Ribose- Arabi- MN_ phosphate myces phosphate myces phos- dopsis 105 pyropho- cerevisiae pyrophos- cerevisiae phate thaliana spho- (strain phokinase S288c pyro- (Mouse- kinase ATCC phos- ear 204508 / pho- cress) S288c) kinase (Baker's yeast) ScHIS 160586 P32895 Ribose- Saccharo- P38689 Ribose- Saccharo- MN_ phosphate myces phosphate myces 106 pyrophos- cerevisiae pyrophos- cerevisiae pho- (strain phokinase S288c kinase ATCC 204508 / S288c) (Baker's yeast) ScHIS 157191 P23254 Trans- Saccharo- P32895 Ribose- Saccharo- P38689 Ribose- Saccharo- MN_ ketolase myces phosphate myces phos- myces 107 cerevisiae pyrophos- cerevisiae phate cerevisiae (strain phokinase S288c pyro- S288c ATCC phos- 204508 / pho- S288c) kinase (Baker's yeast) ScHIS 168183 P23254 Trans- Saccharo- Q12265 Ribose- Saccharo- P38620 Ribose- Saccharo- MN_ ketolase myces phosphate myces phos- myces 108 cerevisiae pyrophos- cerevisiae phate cerevisiae (strain phokinase S288c pyro- S288c ATCC phos- 204508 / pho- S288c) kinase (Baker's yeast) ScHIS 125249 P23254 Trans- Saccharo- Q12265 Ribose- Saccharo- Q680A5 Ribose- Arabi- MN_ ketolase myces phosphate myces phos- dopsis 109 cerevisiae pyrophos- cerevisiae phate thaliana (strain phokinase S288c pyro- (Mouse- ATCC phos- ear 204508 / pho- cress) S288c) kinase (Baker's yeast) ScHIS 157653 P23254 Trans- Saccharo- P0A717 Ribose- Escherichia MN_ ketolase myces phosphate coli 110 cerevisiae pyrophos- (strain (strain phokinase K12) ATCC 204508 / S288c) (Baker's yeast) ScHIS 136093 P23254 Trans- Saccharo- P15019 Trans- Saccharo- P0A717 Ribose- Escheri- MN_ ketolase myces aldolase myces phos- chia 111 cerevisiae cerevisiae phate coli (strain S288c pyro- (strain ATCC phos- K12) 204508 / pho- S288c) kinase (Baker's yeast) ScHIS P06775 Histidine Saccharo- MN_ permease myces 112 cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 160417 P00815 trifunctional Saccharo- P40545 5'ProFAR Saccharo- O59667 Bifunc- Schizo- MN_ histidinol myces isomerase myces tional saccharo- 113 dehydro- cerevisiae cerevisiae phos- myces genase/ (strain S288c phoribo- pombe phos- ATCC syl-AMP ATCC phoribo- 204508 / cyclo- 24843 syl-AMP S288c) hydrolase cyclo- (Baker's and hydro- yeast) phos- lase/ phoribo- phos- syl-ATP phoribo- pyrophos- syl-ATP phatase diphos- phatase ScHIS 116907 P06633 Imidazole- Saccharo- P07172 Histidinol- Saccharo- P38635 Histidi- Saccharo- MN_ glycerol- myces phosphate myces nol- myces 114 phosphate cerevisiae amino- cerevisiae phos- cerevisiae dehydratase (strain trans- S288c phatase S288c ATCC ferase 204508 / S288c) (Baker's yeast) ScHIS 131308 MN_ 41 ScHIS 123614 P00815 trifunctional Saccharo- MN_ histidinol myces 73 dehydro- cerevisiae genase/ (strain phos- ATCC phoribo- 204508 / syl-AMP S288c) cyclohydro- (Baker's lase/ yeast) phos- phoribo- syl-ATP diphos- phatase ScHIS 129393 P06633 Imidazole- Saccharo- MN_ glycerol- myces 74 phosphate cerevisiae dehydratase (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 151455 P07172 Histidinol- Saccharo- MN_ phosphate myces 75 aminotrans- cerevisiae ferase (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 138833 P00498 ATP Saccharo- MN_ phosphori- myces 76 bosyl- cerevisiae transferase (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 164217 P40545 5′ProFAR Saccharo- MN_ isomerase myces 77 cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 159871 P33734 Imidazole Saccharo- MN_ glycerol myces 78 phosphate cerevisiae synthase (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 145179 P00942 Triosephos- I170V Saccharo- MN_ phate myces 79 isomerase cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 137192 P00942 Triosephos- I170T Saccharo- MN_ phate myces 80 isomerase cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast) ScHIS 139699 Q9Z472 ATP Coryne- MN_ phosphori- bacterium 81 bosyl- glutamicum transferase (strain ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025) ScHIS 148665 Q9Z472 ATP N215K, Coryne- MN_ phosphori- L231F, bacterium 82 bosyl- T235A glutamicum transferase (strain ATCC 13032 / DSM 20300 / JCM 1318 /LMG 3730 / NCIMB 10025) ScHIS 109350 Q9Z472 ATP N215K, Coryne- P00815 tri- Saccharo- MN_ phosphori- L231F, bacterium functional myces 84 bosyl- T235A glutamicum histidinol cerevisiae transferase (strain dehydro- S288c ATCC genase/ 13032 / phos- DSM phoribo- 20300 / syl-AMP JCM cyclo- 1318 / hydro- LMG lase/ 3730 / phos- NCIMB phoribo- 10025) syl-ATP diphos- phatase ScHIS 144154 Q9Z472 ATP Coryne- P00815 tri- Saccharo- P00942 Triose- I170V Saccharo- MN_ phosphori- bacterium functional myces phos- myces 85 bosyl- glutamicum histidinol cerevisiae phate cerevisiae transferase (strain dehydro- S288c iso- S288c ATCC genase/ merase 13032 / phos- DSM phoribo- 20300 / syl-AMP JCM cyclo- 1318 / hydro- LMG lase/ 3730 / phos- NCIMB phoribo- 10025) syl-ATP diphos- phatase ScHIS 145171 Q9Z472 ATP N215K, Coryne- P00815 tri- Saccharo- P00942 Triose- I170V Saccharo- MN_ phosphori- L231F, bacterium functional myces phos- myces 86 bosyl- T235A glutamicum histidinol cerevisiae phate cerevisiae transferase (strain dehydro- S288c iso- S288c ATCC genase/ merase 13032 / phos- DSM phoribo- 20300 / syl-AMP JCM cyclo- 1318 / hydrolase/ LMG phos- 3730 / phoribo- NCIMB syl-ATP 10025) diphos- phatase ScHIS 166497 Q9Z472 ATP Coryne- P00815 tri- Saccharo- A4QEF2 Glucose- A243T Coryne- MN_ phosphori- bacterium functional myces 6-phos- bacterium 87 bosyl- glutamicum histidinol cerevisiae phate glutami- transferase (strain dehydro- S288c 1- cum ATCC genase/ dehydro- (strain R) 13032 / phos- genase DSM phoribo- 20300 / syl-AMP JCM cyclo- 1318 / hydro- LMG lase/ 3730 / phos- NCIMB phoribo- 10025) syl-ATP diphos- phatase ScHIS 152555 Q9Z472 ATP N215K, Coryne- P00815 tri- Saccharo- A4QEF2 Glucose- A243T Coryne- MN_ phosphori- L231F, bacterium functional myces 6-phos- bacterium 88 bosyl- T235A glutamicum histidinol cerevisiae phate glutami- transferase (strain dehydro- S288c 1- cum ATCC genase/ dehydro- (strain R) 13032 / phos- genase DSM phoribo- 20300 / syl-AMP JCM cyclo- 1318 / hydro- LMG lase/ 3730 / phos- NCIMB phoribo- 10025) syl-ATP diphos- phatase ScHIS 143866 O66000 Histidine Oenococcus MN_ decarboxy- oeni 89 lase (Leuconostoc oenos) ScHIS 124157 A0A0R Histidine Lactobacillus MN_ 1Y874 decarboxy- aviarius 90 lase subsp. aviarius DSM 20655 ScHIS 68849 A0A1H Histidine S9R Pseudo- MN_ 1TEB8 decarboxy- monas 92 lase sp. bs2935 ScHIS 157127 A0A089 Histidine Pseudo- MN_ YPE5 decarboxy- monas 93 lase rhizosphaerae ScHIS 175497 A0A126 Histidine Pseudo- MN_ S6G9 decarboxy- monas putida 94 lase (Arthrobacter siderocap- sulatus) ScHIS 116642 A0A0J6 Histidine Chromo- MN_ KM89 decarboxy- bacterium sp. 95 lase LK1 ScHIS 171681 A0A0A1 Histidine Citrobacter MN_ R6V3 decarboxy- pasteurii 96 lase ScHIS 171393 A0A1W Histidine Chromo- MN_ 0CM88 decarboxy- bacterium 97 lase haemolyticum ScHIS 152065 P00862 Histidine Lactobacillus MN_ decarboxy- sp. (strain 98 lase 30a) ScHIS 148362 A0A0K6 Histidine Lactobacillus MN_ GJ74 decarboxy- reuteri 99 lase Note: E1, E2, and E3 genes were codon-optimized according to modified codon usage for Cg and Sc

Example 2—Host Evaluation for Histamine Production

Histamine production was also tested in two additional hosts, Bacillus subtilus and Yarrowia lipolytica, which were engineered to express the enzymes from the best-performing Corynebacterium glutamicum and Saccharomyces cerevisiae strains.

Host evaluation designs were selected to express 1-3 enzymes and, each design was tested with four different codon optimizations based on the host organisms C. glutamicum, S. cerevisiae, B. subtilis, and Y. lipolytica. The codon optimizations tested were based on the Kazusa codon usage tables tabulated for each host for gene codon optimization (www.kazusa.or.jp/codon/).

Histamine production was demonstrated in Y. lipolytica (FIG. 6) and B. subtilis (FIG. 7) and further improved in C. glutamicum (FIG. 9) and S. cerevisiae (FIG. 8).

In Y. lipolytica (FIG. 6, Table 4, below) the best performing strain produced 505 mg/L histamine and expressed the histidine decarboxylase from Acinetobacter baumannii strain AB0057 (UniProt ID B7I459), where the DNA sequence was codon-optimized for Y. lipolytica, and the ATP phosphoribosyltransferase from S. cerevisiae S288c (UniProt ID P00498), where the DNA sequence was codon optimized for Y. lipolytica. The same two genes were also tested where the DNA sequence was codon-optimized for B. subtilis and S. cerevisiae and the resulting strains produced no histamine titer.

The second best-performing strain in Y. lipolytica also expressed the histidine decarboxylase from Acinetobacter baumannii strain AB0057 (UniProt ID B7I459), where the DNA sequence was codon-optimized for Y. lipolytica, and the ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (UniProt ID P00499), where the DNA was codon optimized for Y. lipolytica. Versions of these two genes were also tested where the DNA sequence was codon optimized for B. subtilis (which produced 0 titer), codon-optimized for S. cerevisiae (which produced 33 micrograms histamine) and codon-optimized using a combined codon table for S. cerevisiae and C. glutamicum (produced 97 mg/L histamine).

The third best-performing strain in Y. lipolytica produced 258 mg/L histamine and expressed the histidine decarboxylase from Chromobacterium sp. LK1 (UniProt ID A0A0J6KM89), where the DNA sequence was codon optimized for Y. lipolytica, and the ATP phosphoribosyltransferase from C. glutamicum ATCC 13032 (UniProt ID Q9Z472) harboring the amino acid substitutions N215K, L231F, T235A (SEQ ID NO: 7), where the DNA sequence was codon-optimized for Y. lipolytica (SEQ ID NO: 64). Versions of these two genes were also tested where the DNA sequences were codon-optimized for S. cerevisiae (SEQ ID NO: 65, 66) or B. subtilis (SEQ ID NO: 67, 68), and these Y. lipolytica strains produced 1.8 mg/L and 0.3 mg/L, respectively. Accordingly, codon-optimization of genes affects expression in Y. lipolytica.

In B. subtilis (FIG. 7, Table 5, below) the best performing strain produced 18 mg/L histamine and expressed the histamine decarboxylase from Lactobacillus sp. (strain 30a) (UniProt ID P00862)(SEQ ID NO: 4) with the ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (UniProt ID P00499)(SEQ ID NO: 5) where the DNA sequence was codon optimized for Bacillus subtilis (SEQ ID NO: 69, 59). The same two genes were also tested where the DNA sequence was codon-optimized for S. cerevisiae (SEQ ID NO: 70, 60) or modified codon usage table for C. glutamicum and S. cerevisiae (SEQ ID NO: 71, 62), and these strains produced 6.7 mg/L or 0 mg/L histamine, respectively.

The host evaluation designs were also tested in S. cerevisiae and C. glutamicum. In S. cerevisiae (FIG. 8, Table Table 6, below) the best-performing strain produced 111 mg/L histamine and expressed the histamine decarboxylase from Chromobacterium sp. LK1 (UniProt ID A0A0J6KM89)(SEQ ID NO: 51) and the ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c (UniProt ID P00498)(SEQ ID NO: 3), where the DNA sequences were codon-optimized for Y. lipolytica (SEQ ID NO: 63, 53). The same two genes were also tested where the DNA sequences were codon optimized for S. cerevisiae (SEQ ID NO: 65, 57) and B. subtilis (SEQ ID NO: 67, 55) produced 86 mg/L and 101 mg/L, respectively.

In C. glutamicum (FIG. 9, Table 7), the best-performing strain produced 68 mg/L histamine and expressed the histamine decarboxylase from Acinetobacter baumannii (strain AB0057) (UniProt ID B7I459) (SEQ ID NO: 1) with the ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c (UniProt ID P00498) (SEQ ID NO: 3) where the DNA sequences were codon-optimized using a modified codon usage table for C. glutamicum and S. cerevisiae (SEQ ID NO: 72, 73). The same two genes were also tested where the DNA sequence was codon-optimized for Y. lipolytica (SEQ ID NO: 52, 53) or S. cerevisiae (SEQ ID NO: 56, 57), and these strains produced 16 mg/L and 18 microgram/L histamine, respectively.

The second best-performing strain in C. glutamicum produced 15 mg/L histamine and also expressed a histidine decarboxylase from Acinetobacter baumannii strain AB0057 (UniProt ID B7I459) (SEQ ID NO: 1), where the DNA sequence was codon optimized for Y. lipolytica (SEQ ID NO: 52), and an ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (UniProt ID P00499) (SEQ ID NO: 5), where the DNA was codon optimized for Y. lipolytica (SEQ ID NO: 58). These same two genes were also tested, where the DNA sequences were codon-optimized for B. subtilis (SEQ ID NO: 54, 59) (which produced 8 mg/L histamine) or codon-optimized for S. cerevisiae (SEQ ID NO: 56, 60)(which produced 9.3 mg/L histamine).

Since the best performing strain is in the host Y. lipolytica, further strain improvements can be pursued in this host organism. Designs that can further enhance histamine production in Y. lipolytica include:

1. Transketolase (EC 2.2.1.1) (SEQ ID NO: 27), which catalyzes the interconversion of sugars in the pentose phosphate pathway and produces ribose-5-phosphate, which is a precursor to PPRP, the initial metabolite in the histidine biosynthesis pathway.

2. Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) (SEQ ID NO: 28).

3. ATP phosphoribosyltransferase (EC 2.4.2.17) (SEQ ID NO: 5).

4. Trifunctional histidinol dehydrogenase (EC 1.1.1.23)/phosphoribosyl-AMP cyclohydrolase (EC 3.5.4.19)/phosphoribosyl-ATP diphosphatase (EC 3.6.1.31) (SEQ ID NO: 20).

5. Histidinol-phosphate aminotransferase (EC 2.6.1.9) (SEQ ID NO: 14).

6. 5′ProFAR isomerase (EC 5.3.1.16) (SEQ ID NO: 31).

7. Imidazole glycerol phosphate synthase (EC 4.3.1.B2) (SEQ ID NO: 21).

8. Triose-phosphate isomerase (EC 5.3.1.1) harboring the amino acid substitution I170V (SEQ ID NO: 32).

9. Glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49) harboring the amino acid substitution A243T (SEQ ID NO: 26).

10. Various histidine decarboxylases:

-   -   a. UniProt ID A0A089YPE5 (SEQ ID NO: 33)     -   b. UniProt ID A0A126S6G9 (SEQ ID NO: 34)     -   c. UniProt ID A0A0A1R6V3 (SEQ ID NO: 35)     -   d. UniProt ID A0A1W0CM88 (SEQ ID NO: 36)     -   e. UniProt ID P00862 (SEQ ID NO: 4)     -   f. UniProt ID A0A0K6GJ74 (SEQ ID NO: 37)     -   g. UniProt ID TOQL99 (SEQ ID NO: 38)     -   h. UniProt ID A0A1B8HLR1 (SEQ ID NO: 39)

Example 3—Improvement of Histamine Production in Yarrowia lipolytica Engineered to Produce Histamine

Three improvement rounds of genetic engineering were carried out in Yarrowia lipolytica.

First-Improvement Round Genetic Engineering in Yarrowia lipolytica

Strategy: Improve flux into histidine and then histamine by overexpression of two enzymes.

UniProt Codon Enzyme Name ID Organism Description optmization Mutation Histidine B71459 Acinetobacter Last decarboxylation Yarrowia None decarboxylase baumannii step of histamine lypolytica (HDC) biosynthesis ATP P00498 Saccharomyces Upstream step of Yarrowia None phosphoribosyltransferase cerevisiae histidine biosynthesis. lypolytica (ATP-PRase) Utilization of ATP to covert PRPP to PR-ATP

Summary: ATP phosphoribosyltransferase catalyzes the first committed step of histidine biosynthesis pathway. This enzyme would be allosterically feedback-inhibited by histidine and competitively inhibited by AMP and ADP. The results did not indicate activity and/or inhibition of P00498.

Second-Improvement Round Genetic Engineering in Yarrowia lipolytica

Strategy: Overexpression of one enzyme. The final step of histamine biosynthesis was enhanced by utilizing the best first-round histidine decarboxylase which was modified to include a solubility tag to improve protein folding.

UniProt Codon Enzyme Name ID Organism Description optmization Mutation Histidine B71459 Acinetobacter Last decarboxylation Yarrowia None decarboxylase baumannii step of histamine lypolytica (HDC) biosynthesis

Summary: The histidine decarboxylase used for the second round of genetic engineering was the same as for the first round, although the codon optimization was different. Furthermore, an N-terminal solubility tag (MQYKLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFT VT, SEQ ID NO:142) was included in the second-round enzyme.

Third-Improvement Round Genetic Engineering in Yarrowia lipolytica

Strategy: Overexpression of two enzymes in pathways upstream of histidine biosynthesis to improve flux into phosphoribosyl pyrophosphate (PRPP).

UniProt Codon Enzyme Name ID Organism Description optmization Mutation Ribose-phosphate E7EAU9 Bacillus ATP dependent step for Yarrowia L135I pyrophosphokinase amyloliquefaciens synthesis of PRPP lypolytica (RPPK) Glocose-6-phosphate A4QEF2 Corynebacterium Upstream pathway to Yarrowia A243T 1-dehydrogenase glutamicum push carbon flux into lypolytica (G6PDH) ribose-5-phosphate

Summary: Ribose-phosphate pyrophosphokinase is competitively inhibited ADP. The L135I mutation at the ATP binding site on the enzyme relieves ADP inhibition. This strain expressed histamine at a titer of 1.68 g/L of culture medium.

TABLE 4 First-round results for histamine production in Yarrowia lipolytica E1 Enzyme 1 Enzyme 1 E1 Codon E2 Enzyme 2 E2 Enzyme 2 E2 Codon Strain Titer Uniprot activity source Optimization Uniprot activity Modifi- source Optimization name (μg/L) ID name organism Abbrev. ID name cations organism Abbrev. Yarrowia lipolytica YIHISMN_    0 B71459 Histidine Acinetobacter Bacillus P00498 ATP Saccharomyces Bacillus 01 decarboxylase baumannii subtilis phosphoribosyl- cerevisiae subtilis (strain transferase S288c AB0057) YIHISMN_    0 B71459 Histidine Acinetobacter Saccharo- P00498 ATP Saccharomyces Saccharo- 02 decarboxylase baumannii myces phosphoribosyl- cerevisiae myces (strain cerevisiae transferase S288c cerevisiae AB0057) YIHISMN_ 505019 B71459 Histidine Acinetobacter Yarrowia P00498 ATP Saccharomyces Yarrowia 03 decarboxylase baumannii lipolytica phosphoribosyl- cerevisiae lipolytica (strain transferase S288c AB0057) YIHISMN_    0 P00862 Histidine Lactobacillus Bacillus P00499 ATP Salmonella Bacillus 04 decarboxylase sp. subtilis phosphoribosyl- typhimurium subtilis (strain 30a) transferase (strain LT2/ SGSC1412/ ATCC 700720) YIHISMN_  32011 P00862 Histidine Lactobacillus Saccharo- P00499 ATP Salmonella Saccharo- 05 decarboxylase sp. myces phosphoribosyl- typhimurium myces (strain 30a) cerevisiae transferase (strain LT2/ cerevisiae SGSC1412/ ATCC 700720) YIHISMN_   833 P00862 Histidine Lactobacillus Yarrowia P00499 ATP Salmonella Yarrowia 06 decarboxylase sp. lipolytica phosphoribosyl- typhimurium lipolytica (strain 30a) transferase (strain LT2/ SGSC1412/ ATCC 700720) YIHISMN_   299 A0A0J6 Histidine Chromo- Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 07 KM89 decarboxylase bacterium subtilis phosphoribosyl- L231F, glutamicum subtilis sp. LK1 transferase T235A ATCC 13032 YIHISMN_  1778 A0A0J6 Histidine Chromo- Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 08 KM89 decarboxylase bacterium myces phosphoribosyl- L231F, glutamicum myces sp. LK1 cerevisiae transferase T235A ATCC 13032 cerevisiae YIHISMN_ 257949 A0A0J6 Histidine Chromo- Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 09 KM89 decarboxylase bacterium lipolytica phosphoribosyl- L231F, glutamicum lipolytica sp. LK1 transferase T235A ATCC 13032 YIHISMN_    0 B71459 Histidine Acinetobacter Bacillus P00499 ATP Salmonella Bacillus 10 decarboxylase baumannii subtilis phosphoribosyl- typhimurium subtilis (strain transferase LT2 AB0057) YIHISMN_  96836 B71459 Histidine Acinetobacter modified P00499 ATP Salmonella modified 11 decarboxylase baumannii codon phosphoribosyl- typhimurium codon (strain usage for transferase LT2 usage for AB0057) Cg and Sc Cg and Sc YIHISMN_   33 B71459 Histidine Acinetobacter Saccharo- P00499 ATP Salmonella Saccharo- 12 decarboxylase baumannii myces phosphoribosyl- typhimurium myces (strain cerevisiae transferase LT2 cerevisiae AB0057) YIHISMN_ 366139 B71459 Histidine Acinetobacter Yarrowia P00499 ATP Salmonella Yarrowia 13 decarboxylase baumannii lipolytica phosphoribosyl- typhimurium lipolytica (strain transferase LT2 AB0057) YIHISMN_   23 P00862 Histidine Lactobacillus Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 14 decarboxylase sp. subtilis phosphoribosyl- L231F, glutamicum subtilis (strain 30a) transferase T235A ATCC 13032 YIHISMN_   26 P00862 Histidine Lactobacillus modified Q9Z472 ATP N215K, Corynebacterium modified 15 decarboxylase sp. codon phosphoribosyl L231F, glutamicum codon (strain 30a) usage for transferase T235A ATCC 13032 usage for Cg and Sc Cg and Sc YIHISMN_   56 P00862 Histidine Lactobacillus Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 16 decarboxylase sp. myces phosphoribosyl- L231F, glutamicum myces (strain 30a) cerevisiae transferase T235A ATCC 13032 cerevisiae YIHISMN_  1406 P00862 Histidine Lactobacillus Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 17 decarboxylase sp. lipolytica phosphoribosyl- L231F, glutamicum lipolytica (strain 30a) transferase T235A ATCC 13032 YIHISMN_ A0A0J6 Histidine Chromo- Bacillus P00498 ATP Saccharomyces Bacillus 18 KM89 decarboxylase bacterium subtilis phosphoribosyl- cerevisiae subtilis sp. LK1 transferase S288c YIHISMN_  90046 A0A0J6 Histidine Chromo- modified P00498 ATP Saccharomyces modified 19 KM89 decarboxylase bacterium codon phosphoribosyl- cerevisiae codon sp. LK1 usage for transferase S288c usage for Cg and Sc Cg and Sc YIHISMN_  1639 A0A0J6 Histidine Chromo- Saccharo- P00498 ATP Saccharomyces Saccharo- 20 KM89 decarboxylase bacterium myces phosphoribosyl- cerevisiae myces sp. LK1 cerevisiae transferase S288c cerevisiae

TABLE 5 First-round results for production of histamine in Bacillus subtilis E1 Enzyme 1 Enzyme 1 E1 Codon E2 Enzyme 2 E2 Enzyme 2 E2 Codon Strain Titer Uniprot activity source Optimization Uniprot activity Modifi- source Optimization name (μg/L) ID name organism Abbrev. ID name cations organism Abbrev. BsHISMN_ B71459 Histidine Acinetobacter Yarrowia P00498 ATP Saccharomyces Yarrowia 01 decarboxylase baumannii lipolytica phosphoribosyl- cerevisiae lipolytica (strain transferase S288c AB0057) BsHISMN_ 919.7 P00862 Histidine Lactobacillus Yarrowia P00499 ATP Salmonella Yarrowia 02 decarboxylase sp. lipolytica phosphoribosyl- typhimurium lipolytica (strain 30a) transferase LT2 BsHISMN_ 2.4 A0A0J6 Histidine Chromo- modified Q9Z472 ATP N215K, Corynebacterium modified 03 KM89 decarboxylase bacterium codon phosphoribosyl- L231F, glutamicum codon sp. LK1 usage for transferase T235A ATCC 13032 usage for Cg and Sc Cg and Sc BsHISMN_ 9156.1 P00862 Histidine Lactobacillus Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 04 decarboxylase sp. subtilis phosphoribosyl- L231F, glutamicum subtilis (strain 30a) transferase T235A ATCC 13032 BsHISMN_ 5057.2 P00862 Histidine Lactobacillus modified Q9Z472 ATP N215K, Corynebacterium modified 05 decarboxylase sp. codon phosphoribosyl L231F, glutamicum codon (strain 30a) usage for transferase T235A ATCC 13032 usage for Cg and Sc Cg and Sc BsHISMN_ P00862 Histidine Lactobacillus Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 06 decarboxylase sp. lipolytica phosphoribosyl- L231F, glutamicum lipolytica (strain 30a) transferase T235A ATCC 13032 BsHISMN_ 2532.4 B71459 Histidine Acinetobacter Bacillus P00498 ATP Saccharomyces Bacillus 07 decarboxylase baumannii subtilis phosphoribosyl- cerevisiae subtilis (strain transferase S288c AB0057) BsHISMN_ 13183.4 B71459 Histidine Acinetobacter modified P00498 ATP Saccharomyces modified 08 decarboxylase baumannii codon phosphoribosyl- cerevisiae codon (strain usage for transferase S288c usage for AB0057) Cg and Sc Cg and Sc BsHISMN_ 114.3 B71459 Histidine Acinetobacter Saccharo- P00498 ATP Saccharomyces Saccharo- 09 decarboxylase baumannii myces phosphoribosyl- cerevisiae myces (strain cerevisiae transferase S288c cerevisiae AB0057) BsHISMN_ 18336.5 P00862 Histidine Lactobacillus Bacillus P00499 ATP Salmonella Bacillus 10 decarboxylase sp. subtilis phosphoribosyl- typhimurium subtilis (strain 30a) transferase LT2 BsHISMN_ 0 P00862 Histidine Lactobacillus modified P00499 ATP Salmonella modified 11 decarboxylase sp. codon phosphoribosyl- typhimurium codon (strain 30a) usage for transferase LT2 usage for Cg and Sc Cg and Sc BsHISMN_ 6778.2 P00862 Histidine Lactobacillus Saccharo- P00499 ATP Salmonella Saccharo- 12 decarboxylase sp. myces phosphoribosyl- typhimurium myces (strain 30a) cerevisiae transferase LT2 cerevisiae BsHISMN_ A0A0J6 Histidine Chromo- Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 13 KM89 decarboxylase bacterium subtilis phosphoribosyl- L231F, glutamicum subtilis sp. LK1 transferase T235A ATCC 13032 BsHISMN_ 1071.1 A0A0J6 Histidine Chromo- Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 14 KM89 decarboxylase bacterium myces phosphoribosyl- L231F, glutamicum myces sp. LK1 cerevisiae transferase T235A ATCC 13032 cerevisiae BsHISMN_ A0A0J6 Histidine Chromo- Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 15 KM89 decarboxylase bacterium lipolytica phosphoribosyl- L231F, glutamicum lipolytica sp. LK1 transferase T235A ATCC 13032 BsHISMN_ 233.4 B71459 Histidine Acinetobacter Bacillus P00499 ATP Salmonella Bacillus 16 decarboxylase baumannii subtilis phosphoribosyl- typhimurium subtilis (strain transferase LT2 AB0057) BsHISMN_ 16.2 B71459 Histidine Acinetobacter modified P00499 ATP Salmonella modified 17 decarboxylase baumannii codon phosphoribosyl- typhimurium codon (strain usage for transferase LT2 usage for AB0057) Cg and Sc Cg and Sc BsHISMN_ 61 B71459 Histidine Acinetobacter Saccharo- P00499 ATP Salmonella Saccharo- 18 decarboxylase baumannii myces phosphoribosyl- typhimurium myces (strain cerevisiae transferase LT2 cerevisiae AB0057) BsHISMN_ 1413.5 B71459 Histidine Acinetobacter Yarrowia P00499 ATP Salmonella Yarrowia 19 decarboxylase baumannii lipolytica phosphoribosyl- typhimurium lipolytica (strain transferase LT2 AB0057) BsHISMN_ 6630.6 P00862 Histidine Lactobacillus Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 20 decarboxylase sp. myces phosphoribosyl- L231F, glutamicum myces (strain 30a) cerevisiae transferase T235A ATCC 13032 cerevisiae BsHISMN_ 43.8 A0A0J6 Histidine Chromo- Bacillus P00498 ATP Saccharomyces Bacillus 21 KM89 decarboxylase bacterium subtilis phosphoribosyl- cerevisiae subtilis sp. LK1 transferase S288c BsHISMN_ A0A0J6 Histidine Chromo- modified P00498 ATP Saccharomyces modified 22 KM89 decarboxylase bacterium codon phosphoribosyl- cerevisiae codon sp. LK1 usage for transferase S288c usage for Cg and Sc Cg and Sc BsHISMN_ 529 A0A0J6 Histidine Chromo- Saccharo- P00498 ATP Saccharomyces Saccharo- 23 KM89 decarboxylase bacterium myces phosphoribosyl- cerevisiae myces sp. LK1 cerevisiae transferase 5288c cerevisiae BsHISMN_ 15026.1 A0A0J6 Histidine Chromo- Yarrowia P00498 ATP Saccharomyces Yarrowia 24 KM89 decarboxylase bacterium lipolytica phosphoribosyl- cerevisiae lipolytica sp. LK1 transferase S288c BsHISMN_ A0A0J6 Histidine Chromo- modified Q9Z472 ATP Corynebacterium modified 25 KM89 decarboxylase bacterium codon phosphoribosyl- glutamicum codon sp. LK1 usage for transferase ATCC 13032 usage for Cg and Sc Cg and Sc

TABLE 6 Host evaluation designs for production of histamine tested in Saccharomyces cerevisiae E1 Enzyme 1 Enzyme 1 E1 Codon E2 Enzyme 2 E2 Enzyme 2 E2 Codon Strain Titer Uniprot activity source Optimization Uniprot activity Modifi- source Optimization name (μg/L) ID name organism Abbrev. ID name cations organism Abbrev. Saccharomyces cerevisiae ScHISMN_  17466 P00862 Histidine Lactobacillus Bacillus P00499 ATP Salmonella Bacillus 116 decarboxylase sp. subtilis phosphoribosyl- typhimurium subtilis (strain 30a) transferase LT2 ScHISMN_  28646 P00862 Histidine Lactobacillus Saccharo- P00499 ATP Salmonella Saccharo- 117 decarboxylase sp. myces phosphoribosyl- typhimurium myces (strain 30a) cerevisiae transferase LT2 cerevisiae ScHISMN_  48150 P00862 Histidine Lactobacillus Yarrowia P00499 ATP Salmonella Yarrowia 118 decarboxylase sp. lipolytica phosphoribosyl- typhimurium lipolytica (strain 30a) transferase LT2 ScHISMN_  59265 A0A0J6 Histidine Chromo- Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 119 KM89 decarboxylase bacterium subtilis phosphoribosyl- L231F, glutamicum subtilis sp. LK1 transferase T235A ATCC 13032 ScHISMN_  72566 A0A0J6 Histidine Chromo- modified Q9Z472 ATP N215K, Corynebacterium modified 120 KM89 decarboxylase bacterium codon phosphoribosyl L231F, glutamicum codon sp. LK1 usage for transferase T235A ATCC 13032 usage for Cg and Sc Cg and Sc ScHISMN_  46418 A0A0J6 Histidine Chromo- Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 121 KM89 decarboxylase bacterium myces phosphoribosyl- L231F, glutamicum myces sp. LK1 cerevisiae transferase T235A ATCC 13032 cerevisiae ScHISMN_  64087 A0A0J6 Histidine Chromo- Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 122 KM89 decarboxylase bacterium lipolytica phosphoribosyl- L231F, glutamicum lipolytica sp. LK1 transferase T235A ATCC 13032 ScHISMN_  80704 B71459 Histidine Acinetobacter Bacillus P00499 ATP Salmonella Bacillus 123 decarboxylase baumannii subtilis phosphoribosyl- typhimurium subtilis (strain transferase LT2 AB0057) ScHISMN_  70043 B71459 Histidine Acinetobacter Yarrowia P00499 ATP Salmonella Yarrowia 124 decarboxylase baumannii lipolytica phosphoribosyl- typhimurium lipolytica (strain transferase LT2 AB0057) ScHISMN_  25331 P00862 Histidine Lactobacillus Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 125 decarboxylase sp. subtilis phosphoribosyl- L231F, glutamicum subtilis (strain 30a) transferase T235A ATCC 13032 ScHISMN_  33970 P00862 Histidine Lactobacillus modified Q9Z472 ATP N215K, Corynebacterium modified 126 decarboxylase sp. codon phosphoribosyl L231F, glutamicum codon (strain 30a) usage for transferase T235A ATCC 13032 usage for Cg and Sc Cg and Sc ScHISMN_  21402 P00862 Histidine Lactobacillus Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 127 decarboxylase sp. myces phosphoribosyl- L231F, glutamicum myces (strain 30a) cerevisiae transferase T235A ATCC 13032 cerevisiae ScHISMN_  41854 P00862 Histidine Lactobacillus Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 128 decarboxylase sp. lipolytica phosphoribosyl- L231F, glutamicum lipolytica (strain 30a) transferase T235A ATCC 13032 ScHISMN_ 101496 A0A0J6 Histidine Chromo- Bacillus P00498 ATP Saccharomyces Bacillus 129 KM89 decarboxylase bacterium subtilis phosphoribosyl- cerevisiae subtilis sp. LK1 transferase S288c ScHISMN_  85546 A0A0J6 Histidine Chromo- Saccharo- P00498 ATP Saccharomyces Saccharo- 130 KM89 decarboxylase bacterium myces phosphoribosyl- cerevisiae myces sp. LK1 cerevisiae transferase S288c cerevisiae ScHISMN_ 111109 A0A0J6 Histidine Chromo- Yarrowia P00498 ATP Saccharomyces Yarrowia 131 KM89 decarboxylase bacterium lipolytica phosphoribosyl- cerevisiae lipolytica sp. LK1 transferase S288c

TABLE 7 Host evaluation designs for production of histamine tested in Corynebacterium glutamicum E1 Enzyme 1 Enzyme 1 E1 Codon E2 Enzyme 2 E2 Enzyme 2 E2 Codon Strain Titer Uniprot activity source Optimization Uniprot activity Modifi- source Optimization name (μg/L) ID name organism Abbrev. ID name cations organism Abbrev. CgHISMN_ B71459 Histidine Acinetobacter Bacillus P00498 ATP Saccharomyces Bacillus 70 decarboxylase baumannii subtilis phosphoribosyl- cerevisiae subtilis (strain transferase S288c AB0057) CgHISMN_ 68395.9 B71459 Histidine Acinetobacter modified P00498 ATP Saccharomyces modified 71 decarboxylase baumannii codon phosphoribosyl- cerevisiae codon (strain usage for transferase S288c usage for AB0057) Cg and Sc Cg and Sc CgHISMN_ 18 B71459 Histidine Acinetobacter Saccharo- P00498 ATP Saccharomyces Saccharo- 72 decarboxylase baumannii myces phosphoribosyl- cerevisiae myces (strain cerevisiae transferase S288c cerevisiae AB0057) CgHISMN_ 16325.5 B71459 Histidine Acinetobacter Yarrowia P00498 ATP Saccharomyces Yarrowia 73 decarboxylase baumannii lipolytica phosphoribosyl- cerevisiae lipolytica (strain transferase S288c AB0057) CgHISMN_ 4883.6 P00862 Histidine Lactobacillus Bacillus P00499 ATP Salmonella Bacillus 74 decarboxylase sp. subtilis phosphoribosyl- typhimurium subtilis (strain 30a) transferase LT2 CgHISMN_ P00862 Histidine Lactobacillus Saccharo- P00499 ATP Salmonella Saccharo- 75 decarboxylase sp. myces phosphoribosyl- typhimurium myces (strain 30a) cerevisiae transferase LT2 cerevisiae CgHISMN_ P00862 Histidine Lactobacillus Yarrowia P00499 ATP Salmonella Yarrowia 76 decarboxylase sp. lipolytica phosphoribosyl- typhimurium lipolytica (strain 30a) transferase LT2 CgHISMN_ 5.4 A0A0J6 Histidine Chromo- Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 77 KM89 decarboxylase bacterium subtilis phosphoribosyl- L231F, glutamicum subtilis sp. LK1 transferase T235A ATCC 13032 CgHISMN_ 88.6 A0A0J6 Histidine Chromo- Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 78 KM89 decarboxylase bacterium myces phosphoribosyl- L231F, glutamicum myces sp. LK1 cerevisiae transferase T235A ATCC 13032 cerevisiae CgHISMN_ A0A0J6 Histidine Chromo- Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 79 KM89 decarboxylase bacterium lipolytica phosphoribosyl- L231F, glutamicum lipolytica sp. LK1 transferase T235A ATCC 13032 CgHISMN_ 8368.2 B71459 Histidine Acinetobacter Bacillus P00499 ATP Salmonella Bacillus 80 decarboxylase baumannii subtilis phosphoribosyl- typhimurium subtilis (strain LT2 AB0057) CgHISMN_ 9.3 B71459 Histidine Acinetobacter Saccharo- P00499 ATP Salmonella Saccharo- 81 decarboxylase baumannii myces phosphoribosyl- typhimurium myces (strain cerevisiae transferase LT2 cerevisiae AB0057) CgHISMN_ 15529.4 B71459 Histidine Acinetobacter Yarrowia P00499 ATP Salmonella Yarrowia 82 decarboxylase baumannii lipolytica phosphoribosyl- typhimurium lipolytica (strain transferase LT2 AB0057) CgHISMN_ P00862 Histidine Lactobacillus Bacillus Q9Z472 ATP N215K, Corynebacterium Bacillus 83 decarboxylase sp. subtilis phosphoribosyl- L231F, glutamicum subtilis (strain 30a) transferase T235A ATCC 13032 CgHISMN_ 2.6 P00862 Histidine Lactobacillus Saccharo- Q9Z472 ATP N215K, Corynebacterium Saccharo- 84 decarboxylase sp. myces phosphoribosyl- L231F, glutamicum myces (strain 30a) cerevisiae transferase T235A ATCC 13032 cerevisiae CgHISMN_ 6134 P00862 Histidine Lactobacillus Yarrowia Q9Z472 ATP N215K, Corynebacterium Yarrowia 85 decarboxylase sp. lipolytica phosphoribosyl- L231F, glutamicum lipolytica (strain 30a) transferase T235A ATCC 13032 CgHISMN_ 197 A0A0J6 Histidine Chromo- Bacillus P00498 ATP Saccharomyces Bacillus 86 KM89 decarboxylase bacterium subtilis phosphoribosyl- cerevisiae subtilis sp. LK1 transferase S288c

TABLE 8 SEQ ID NO Cross-Reference Table SEQ ID Sequence Type with Uniprot Codon NO Modifications ID Activity name Source organism Optimization Abbrev.  1 AA seq for B71459 histidine decarboxylase Acinetobacter baumannii enzyme B71459 (strain AB0057)  2 AA seq for Q9KJU3 Imidazoleglycerol- Corynebacterium glutamicum enzyme Q9KJU3 phosphate dehydratase  3 AA seq for P00498 ATP phosphoribosyltransferase Saccharomyces cerevisiae enzyme P00498  4 AA seq for P00862 histidine decarboxylase Lactobacillus sp. (strain 30a) enzyme P00862  5 AA seq for P00499 ATP phosphoribosyltransferase Salmonella typhimurium enzyme P00499 with (strain LT2/SGSC1412/ deletion of Q207-E208 ATCC 700720)  6 AA seq for J6KM89 histidine decarboxylase Chromobacterium sp. LK1 enzyme J6KM89  7 AA seq for Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum enzyme Q9Z472 (strain ATCC 13032/ with substitution DSM 20300/JCM 1318/ N215K, L231F, T235A LMG 3730/NCIMB 10025)  8 AA seq for E3QMN8 histidine decarboxylase Methanosarcina barkeri enzyme E3QMN8 str. Wiesmoor  9 AA seq for Q467R8 histidine decarboxylase Methanosarcina barkeri enzyme Q467R8 (strain Fusaro/DSM 804)  10 AA seq for Q05733 histidine decarboxylase Drosophila melanogaster enzyme Q05733  11 AA seq for P54772 histidine decarboxylase Solanum lycopersicum enzyme P54772  12 AA seq for P23738 histidine decarboxylase Mus musculus enzyme P23738  13 AA seq for O68602 1-(5-phosphoribosyl)5[(5- Corynebacterium glutamicum enzyme O68602 phosphoribosylamino) methylideneamino] imidazole-4- carboxamide isomerase  14 AA seq for Q9KJU4 Histidinol-phosphate Corynebacterium glutamicum enzyme Q9KJU4 aminotransferase  15 AA seq for Q8NNT5 Histidinol dehydrogenase Corynebacterium glutamicum enzyme Q8NNT5  16 AA seq for Q9Z471 Phosphoribosyl- Corynebacterium glutamicum enzyme Q9Z471 ATP pyrophosphatase  17 AA seq for O31139 Imidazole glycerol phosphate Corynebacterium glutamicum enzyme O31139 synthase subunit  18 AA seq for O69043 Imidazole glycerol phosphate Corynebacterium glutamicum enzyme O69043 synthase subunit  19 AA seq for Q8NNT9 phosphoribosyl- Corynebacterium glutamicum enzyme Q8NNT9 AMP cyclohydrolase  22 AA seq for Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum enzyme Q9Z472 (strain ATCC 13032/ DSM 20300/JCM 1318/ LMG 3730/NCIMB 10025)  20 AA seq for P00815 histidinol dehydrogenase, Saccharomyces cerevisiae enzyme P00815 phosphoribosyl- AMP cyclohydrolase, phosphoribosyl- ATP diphosphatase  21 AA seq for P33734 Imidazole glycerol phosphate Saccharomyces cerevisiae enzyme P33734 synthase subunit HisF  23 AA seq for P07172 histidinol- Saccharomyces cerevisiae enzyme P07172 phosphate transaminase  24 AA seq for P06633 Imidazoleglycerol- Saccharomyces cerevisiae enzyme P06633 phosphate dehydratase  25 AA seq for P38635 histidinol-phosphatase Saccharomyces cerevisiae enzyme P38635  26 AA seq for A4QEF2 Glucose-6-phosphate Corynebacterium glutamicum enzyme A4QEF2 with 1-dehydrogenase substitution A243T (G6PD) (EC 1.1.1.49) (strain R)  27 AA seq for P23254 Transketolase Saccharomyces cerevisiae enzyme P23254 (strain ATCC 204508/S288c) (Baker's yeast)  28 AA seq for Q12265 Ribose-phosphate Saccharomyces cerevisiae enzyme Q12265 pyrophosphokinase 5 (strain ATCC 204508/S288c) (EC 2.7.6.1) (Baker's yeast) (Phosphoribosyl pyrophosphate synthase 5)  30 DNA seq1 for Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum native enzyme Q9Z472  29 DNA seq1 for P00815 histidinol dehydrogenase, Saccharomyces cerevisiae native enzyme P00815 phosphoribosyl- AMP cyclohydrolase, phosphoribosyl- ATP diphosphatase  31 AA seq for P40545 1-(5-phosphoribosyl)-5-[(5- Saccharomyces cerevisiae enzyme P40545 phosphoribosylamino) (strain ATCC 204508/S288c) methylideneamino] (Baker's yeast) imidazole-4-carboxamide isomerase (EC 5.3.1.16) (5-proFAR isomerase) (Phosphoribosylformimino-5- aminoimidazole carboxamide ribotide isomerase)  32 AA seq for P00942 Triosephosphate isomerase Saccharomyces cerevisiae enzyme P00942 with (TIM) (EC 5.3.1.1) (strain ATCC 204508/S288c) substitution 1170V (Triose-phosphate isomerase) (Baker's yeast)  33 AA seq for enzyme A0A089 Histidine decarboxylase Pseudomonas rhizosphaerae A0A089YPE5 YPE5 (HDC) (EC 4.1.1.22)  34 AA seq for enzyme A0A126 Histidine decarboxylase Pseudomonas putida AOA12656G9 56G9 (HDC) (EC 4.1.1.22) (Arthrobacter siderocapsulatus)  35 AA seq for enzyme A0A0A1 Histidine decarboxylase Citrobacter pasteurii A0A0A1R6V3 R6V3 (HDC) (EC 4.1.1.22)  36 AA seq for enzyme A0A1W0 Histidine decarboxylase Chromobacterium haemolyticum A0A1W0CM88 CM88 (HDC) (EC 4.1.1.22)  37 AA seq for enzyme A0A0K6 Histidine decarboxylase Lactobacillus reuteri A0A0K6GJ74 GJ74 proenzyme  38 AA seq for T0QL99 Histidine decarboxylase Aeromonas salmonicida enzyme T0QL99 (EC 4.1.1.22) (Fragment) subsp. pectinolytica 34mel  39 AA seq for enzyme A0A1B8 Histidine decarboxylase Morganella psychrotolerans A0A1B8HLR1 HLR1 (HDC) (EC 4.1.1.22)  40 AA seq for enzyme A0A0C1 Histidine decarboxylase Lactobacillus fructivorans A0A0C1PR48 PR48 proenzyme  41 AA seq for P0A717 Ribose-phosphate Escherichia coli (strain K12) enzyme P0A717 pyrophosphokinase (RPPK) (EC 2.7.6.1) (5-phospho-D-ribosyl alpha-1-diphosphate) (Phosphoribosyl diphosphate synthase) (Phosphoribosyl pyrophosphate synthase) (P-Rib-PP synthase) (PRPP synthase) (PRPPase)  42 AA seq for Q680A5 Ribose-phosphate Arabidopsis thaliana enzyme Q680A5 pyrophosphokinase 4 (Mouse-ear cress) (EC 2.7.6.1) (Phosphoribosyl pyrophosphate synthase 4)  43 AA seq for P38620 Ribose-phosphate Saccharomyces cerevisiae enzyme P38620 pyrophosphokinase 2 (strain ATCC 204508/S288c) (EC 2.7.6.1) (Baker's yeast) (Phosphoribosyl pyrophosphate synthase 2)  44 AA seq for P32895 Ribose-phosphate Saccharomyces cerevisiae enzyme P32895 pyrophosphokinase 1 (strain ATCC 204508/S288c) (EC 2.7.6.1) (Baker's yeast) (Phosphoribosyl pyrophosphate synthase 1)  45 AA seq for P38689 Ribose-phosphate Saccharomyces cerevisiae enzyme P38689 pyrophosphokinase 3 (strain ATCC 204508/S288c) (EC 2.7.6.1) (Baker's yeast) (Phosphoribosyl pyrophosphate synthase 3)  46 AA seq for P15019 Transaldolase (EC 2.2.1.2) Saccharomyces cerevisiae enzyme P15019 (strain ATCC 204508/S288c) (Baker's yeast)  47 AA seq for P06775 Histidine permease Saccharomyces cerevisiae enzyme P06775 (strain ATCC 204508/S288c) (Baker's yeast)  48 AA seq for O59667 Histidine biosynthesis Schizosaccharomyces pombe enzyme O59667 bifunctional protein (strain 972/ATCC 24843) his7 [Includes: (Fission yeast) Phosphoribosyl- AMP cyclohydrolase (EC 3.5.4.19); Phosphoribosyl- ATP pyrophosphatase (EC 3.6.1.31)]  49 AA seq for O66000 Histidine decarboxylase Oenococcus oeni enzyme O66000 proenzyme (Leuconostoc oenos)  50 AA seq for enzyme A0A0R1 Pyruvoyl family Lactobacillus aviarius subsp. A0A0R1Y874 Y874 histidine decarboxylase aviarius DSM 20655  51 AA seq for enzyme A0A0J6K Histidine decarboxylase Chromobacterium sp. LK1 A0A0J6KM89 M89 (HDC) (EC 4.1.1.22)  52 DNA seq1 for B71459 Histidine decarboxylase Acinetobacter baumannii Yarrowia lipolytica enzyme B71459 (strain AB0057)  53 DNA seq1 for P00498 ATP phosphoribosyltransferase Saccharomyces cerevisiae Yarrowia lipolytica enzyme P00498 (strain ATCC 204508/S288c) (Baker's yeast)  54 DNA seq2 for B71459 Histidine decarboxylase Acinetobacter baumannii Bacillus subtillus enzyme B71459 (strain AB0057)  55 DNA seq2 for P00498 ATP phosphoribosyltransferase Saccharomyces cerevisiae Bacillus subtillus enzyme P00498 (strain ATCC 204508/S288c) (Baker's yeast)  56 DNA seq3 for B71459 Histidine decarboxylase Acinetobacter baumannii Saccharomyces cerevisiae enzyme B71459 (strain AB0057)  57 DNA seq3 for P00498 ATP phosphoribosyltransferase Saccharomyces cerevisiae Saccharomyces cerevisiae enzyme P00498 (strain ATCC 204508/S288c) (Baker's yeast)  58 DNA seq1 for P00499 ATP phosphoribosyltransferase Salmonella typhimurium Yarrowia lipolytica enzyme P00499 with (strain LT2/SGSC1412/ deletion of Q207-E208 ATCC 700720)  59 DNA seq2 for P00499 ATP phosphoribosyltransferase Salmonella typhimurium Bacillus subtillus enzyme P00499 with (strain LT2/SGSC1412/ deletion of Q207-E208 ATCC 700720)  60 DNA seq3 for P00499 ATP phosphoribosyltransferase Salmonella typhimurium Saccharomyces cerevisiae enzyme P00499 with (strain LT2/SGSC1412/ deletion of Q207-E208 ATCC 700720)  61 DNA seq4 for B71459 Histidine decarboxylase Acinetobacter baumannii modified codon usage for enzyme B71459 (HDC) (EC 4.1.1.22) (strain AB0057) Corynebacterium glutamicum and Saccharomyces cerevisiae  62 DNA seq4 for P00499 ATP phosphoribosyltransferase Salmonella typhimurium modified codon usage for enzyme P00499 with (strain LT2/SGSC1412/ Corynebacterium glutamicum and deletion of Q207-E208 ATCC 700720) Saccharomyces cerevisiae  63 DNA seq1 for enzyme A0A0J6K Histidine decarboxylase Chromobacterium sp. LK1 Yarrowia lipolytica A0A0J6KM89 M89  64 DNA seq1 Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum Yarrowia lipolytica for enzyme Q9Z472 (strain ATCC 13032/ with substitution DSM 20300/JCM 1318/ N215K, L231F, T235A LMG 3730/NCIMB 10025)  65 DNA seq2 for enzyme A0A0J6K Histidine decarboxylase Chromobacterium sp. LK1 Saccharomyces cerevisiae A0A0J6KM89 M89  66 DNA seq2 Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum Saccharomyces cerevisiae for enzyme Q9Z472 (strain ATCC 13032/ with substitution DSM 20300/JCM 1318/ N215K, L231F, T235A LMG 3730/NCIMB 10025)  67 DNA seq3 A0A0J6K Histidine decarboxylase Chromobacterium sp. LK1 Bacillus subtillus for enzyme M89 A0A0J6KM89  68 DNA seq3 Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum Bacillus subtillus for enzyme Q9Z472 (strain ATCC 13032/ with substitution DSM 20300/JCM 1318/ N215K, L231F, T235A LMG 3730/NCIMB 10025)  69 DNA seq1 for P00862 Histidine decarboxylase Lactobacillus sp. (strain 30a) Bacillus subtillus enzyme P00862 proenzyme  70 DNA seq2 for P00862 Histidine decarboxylase Lactobacillus sp. (strain 30a) Saccharomyces cerevisiae enzyme P00862 proenzyme  71 DNA seq3 for P00862 Histidine decarboxylase Lactobacillus sp. (strain 30a) modified codon usage for enzyme P00862 proenzyme Corynebacterium glutamicum and Saccharomyces cerevisiae  72 DNA seq5 for B71459 Histidine decarboxylase Acinetobacter baumannii modified codon usage for enzyme B71459 (strain AB0057) Corynebacterium glutamicum and Saccharomyces cerevisiae  73 DNA seq4 for P00498 ATP phosphoribosyltransferase Saccharomyces cerevisiae modified codon usage for enzyme P00498 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (Baker's yeast) Saccharomyces cerevisiae  74 AA seq for enzyme A0A1H1 Histidine decarboxylase Pseudomonas sp. bs2935 modified codon usage for A0A1H1TEB8 TEB8 (HDC) (EC 4.1.1.22) Corynebacterium glutamicum and Saccharomyces cerevisiae  75 DNA seq1 for E3QMN8 histidine decarboxylase Methanosarcina barkeri Corynebacterium glutamicum enzyme E3QMN8 str. Wiesmoor  76 DNA seq1 for Q467R8 histidine decarboxylase Methanosarcina barkeri Corynebacterium glutamicum enzyme Q467R8 (strain Fusaro/DSM 804)  77 DNA seq4 for P00862 histidine decarboxylase Lactobacillus sp. (strain 30a) Corynebacterium glutamicum enzyme P00862  78 DNA seq6 for B71459 histidine decarboxylase Acinetobacter baumannii Corynebacterium glutamicum enzyme B71459 (strain AB0057)  79 DNA seq1 for Q05733 histidine decarboxylase Drosophila melanogaster Corynebacterium glutamicum enzyme Q05733  80 DNA seq1 for J6KM89 histidine decarboxylase Chromobacterium sp. LK1 Corynebacterium glutamicum enzyme J6KM89  81 DNA seq5 for P00499 ATP phosphoribosyltransferase Salmonella typhimurium Corynebacterium glutamicum enzyme P00499 with (strain LT2/SGSC1412/ deletion of Q207-E208 ATCC 700720)  82 DNA seq5 for P00862 histidine decarboxylase Lactobacillus sp. (strain 30a) Saccharomyces cerevisiae enzyme P00862  83 DNA seq for P54772 histidine decarboxylase Solanum lycopersicum Saccharomyces cerevisiae enzyme P54772  84 DNA seq for P23738 histidine decarboxylase Mus musculus Saccharomyces cerevisiae enzyme P23738  85 DNA seq2 for Q05733 histidine decarboxylase Drosophila melanogaster Saccharomyces cerevisiae enzyme Q05733  86 DNA seq2 for J6KM89 histidine decarboxylase Chromobacterium sp. LK1 Saccharomyces cerevisiae enzyme J6KM89  87 DNA seq2 for E3QMN8 histidine decarboxylase Methanosarcina barkeri Saccharomyces cerevisiae enzyme E3QMN8 str. Wiesmoor  88 DNA seq2 for Q467R8 histidine decarboxylase Methanosarcina barkeri Saccharomyces cerevisiae enzyme Q467R8 (strain Fusaro/DSM 804)  89 DNA seq4 Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum Saccharomyces cerevisiae for enzyme Q9Z472 (strain ATCC 13032/ with substitution DSM 20300/JCM 1318/ N215K, L231F, T235A LMG 3730/NCIMB 10025)  90 DNA seq6 for P00499 ATP phosphoribosyltransferase Salmonella typhimurium Saccharomyces cerevisiae enzyme P00499 with (strain LT2/SGSC1412/ deletion of Q207-E208 ATCC 700720)  91 DNA seq for O68602 1-(5-phosphoribosyl)5[(5- Corynebacterium glutamicum native enzyme O68602 phosphoribosylamino) methylideneamino]imidazole- 4-carboxamide isomerase  92 DNA seq for Q9KJU3 Imidazoleglycerol- Corynebacterium glutamicum native enzyme Q9KJU3 phosphate dehydratase  93 DNA seq for Q9KJU4 Histidinol-phosphate Corynebacterium glutamicum native enzyme Q9KJU4 aminotransferase  94 DNA seq for Q8NNT5 Histidinol dehydrogenase Corynebacterium glutamicum native enzyme Q8NNT5  95 DNA seq for Q9Z471 Phosphoribosyl-ATP Corynebacterium glutamicum native enzyme Q9Z471 pyrophosphatase  96 DNA seq for O31139 Imidazole glycerol phosphate Corynebacterium glutamicum native enzyme O31139 synthase subunit  97 DNA seq for O69043 Imidazole glycerol phosphate Corynebacterium glutamicum native enzyme O69043 synthase subunit  98 DNA seq for Q8NNT9 phosphoribosyl- Corynebacterium glutamicum native enzyme Q8NNT9 AMP cyclohydrolase  99 DNA seq5 for P00498 ATP phosphoribosyltransferase Saccharomyces cerevisiae native enzyme P00498 Imidazole glycerol phosphate 100 DNA seq1 for P33734 synthase subunit HisF Saccharomyces cerevisiae native enzyme P33734 101 DNA seq1 for P07172 histidinol-phosphate Saccharomyces cerevisiae native enzyme P07172 transaminase 102 DNA seq for P06633 Imidazoleglycerol- Saccharomyces cerevisiae native enzyme P06633 phosphate dehydratase 103 DNA seq1 for P38635 histidinol-phosphatase Saccharomyces cerevisiae native enzyme P38635 104 DNA seq A0A0C1 Histidine decarboxylase Lactobacillus fructivorans modified codon usage for for enzyme PR48 proenzyme Corynebacterium glutamicum and A0A0C1PR48 Saccharomyces cerevisiae 105 DNA seq for T0QL99 Histidine decarboxylase Aeromonas salmonicida modified codon usage for enzyme T0QL99 (EC 4.1.1.22) (Fragment) subsp. pectinolytica 34mel Corynebacterium glutamicum and Saccharomyces cerevisiae 106 DNA seq A0A1B8 Histidine decarboxylase Morganella psychrotolerans modified codon usage for for enzyme HLR1 (HDC) (EC 4.1.1.22) Corynebacterium glutamicum and A0A1B8HLR1 Saccharomyces cerevisiae 107 DNA seq2 for Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum modified codon usage for enzyme Q9Z472 (ATP-PRT) (ATP-PRTase) (strain ATCC 13032/ Corynebacterium glutamicum and DSM 20300/JCM 1318/ Saccharomyces cerevisiae LMG 3730/NCIMB 10025) 108 DNA seq for P0A717 Ribose-phosphate Escherichia coli (strain K12) modified codon usage for enzyme P0A717 pyrophosphokinase Corynebacterium glutamicum and (RPPK) (EC 2.7.6.1) Saccharomyces cerevisiae (5-phospho-D-ribosyl alpha-1-diphosphate) (Phosphoribosyl diphosphate synthase) (Phosphoribosyl pyrophosphate synthase) (P-Rib-PP synthase) (PRPP synthase) (PRPPase) 109 DNA seq for Q12265 Ribose-phosphate Saccharomyces cerevisiae modified codon usage for enzyme Q12265 pyrophosphokinase 5 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (EC 2.7.6.1) (Phosphoribosyl (Baker's yeast) Saccharomyces cerevisiae pyrophosphate synthase 5) 110 DNA seq for P32895 Ribose-phosphate Saccharomyces cerevisiae modified codon usage for enzyme P32895 pyrophosphokinase 1 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (EC 2.7.6.1) (Phosphoribosyl (Baker's yeast) Saccharomyces cerevisiae pyrophosphate synthase 1) 111 DNA seq for P23254 Transketolase Saccharomyces cerevisiae modified codon usage for enzyme P23254 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (Baker's yeast) Saccharomyces cerevisiae 112 DNA seq for P06775 Histidine permease Saccharomyces cerevisiae modified codon usage for enzyme P06775 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (Baker's yeast) Saccharomyces cerevisiae 113 DNA seq2 for P00815 trifunctional histidinol Saccharomyces cerevisiae modified codon usage for enzyme P00815 dehydrogenase/ (strain ATCC 204508/S288c) Corynebacterium glutamicum and phosphoribosyl-AMP (Baker's yeast) Saccharomyces cerevisiae cyclohydrolase/ phosphoribosyl-ATP diphosphatase 114 DNA seq2 fo P07172 Histidinol-phosphate Saccharomyces cerevisiae modified codon usage for enzyme P07172 aminotransferase (strain ATCC 204508/S288c) Corynebacterium glutamicum and (Baker's yeast) Saccharomyces cerevisiae 115 DNA seq6 for P00498 ATP phosphoribosyltransferase Saccharomyces cerevisiae modified codon usage for enzyme P00498 (ATP-PRT) (ATP-PRTase) (strain ATCC 204508/S288c) Corynebacterium glutamicum and (Baker's yeast) Saccharomyces cerevisiae 116 DNA seq for P40545 1-(5-phosphoribosyl)-5-[(5- Saccharomyces cerevisiae modified codon usage for enzyme P40545 phosphoribosylamino) (strain ATCC 204508/S288c) Corynebacterium glutamicum and methylideneamino] (Baker's yeast) Saccharomyces cerevisiae imidazole-4-carboxamide isomerase (EC 5.3.1.16) (5-proFAR isomerase) (Phosphoribosylformimino- 5-aminoimidazole carboxamide ribotide isomerase) 117 DNA seq2 for P33734 Imidazole Saccharomyces cerevisiae modified codon usage for enzyme P33734 glycerol phosphate (strain ATCC 204508/S288c) Corynebacterium glutamicum and synthase hisHF (Baker's yeast) Saccharomyces cerevisiae 118 DNA seq for P00942 Triosephosphate isomerase Saccharomyces cerevisiae modified codon usage for enzyme P00942 with (TIM) (EC 5.3.1.1) (strain ATCC 204508/S288c) Corynebacterium glutamicum and substitution 1170V (Triose-phosphate isomerase) (Baker's yeast) Saccharomyces cerevisiae 119 DNA seq3 for Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum modified codon usage for enzyme Q9Z472 (ATP-PRT) (ATP-PRTase) (strain ATCC 13032/ Corynebacterium glutamicum and (EC 2.4.2.17) DSM 20300/JCM 1318/ Saccharomyces cerevisiae 120 DNA seq5 Q9Z472 ATP phosphoribosyltransferase LMG 3730/NCIMB 10025) modified codon usage for for enzyme Q9Z472 (ATP-PRT) (ATP-PRTase) (strain ATCC 13032/ Corynebacterium glutamicum and with substitution (EC 2.4.2.17) DSM 20300/JCM 1318/ Saccharomyces cerevisiae N215K, L231F, T235A LMG 3730/NCIMB 10025) 121 DNA seq for O66000 Histidine decarboxylase Oenococcus oeni modified codon usage for enzyme O66000 proenzyme (Leuconostoc oenos) Corynebacterium glutamicum and Saccharomyces cerevisiae 122 DNA seq for enzyme A0A0R1 Pyruvoyl family Lactobacillus aviarius modified codon usage for A0A0R1Y874 Y874 histidine decarboxylase subsp. aviarius DSM 20655 Corynebacterium glutamicum and Saccharomyces cerevisiae 123 DNA seq for enzyme A0A1H1 Histidine decarboxylase Pseudomonas sp. bs2935 modified codon usage for A0A1H1TEB8 with TEB8 (HDC) (EC 4.1.1.22) Corynebacterium glutamicum and substitution S9R Saccharomyces cerevisiae 124 DNA seq for enzyme A0A089 Histidine decarboxylase Pseudomonas rhizosphaerae modified codon usage for A0A089YPE5 YPE5 (HDC) (EC 4.1.1.22) Corynebacterium glutamicum and Saccharomyces cerevisiae 125 DNA seq for enzyme A0A126 Histidine decarboxylase Pseudomonas putida modified codon usage for A0A12656G9 56G9 (HDC) (EC 4.1.1.22) (Arthrobacter siderocapsulatus) Corynebacterium glutamicum and Saccharomyces cerevisiae 126 DNA seq4 for enzyme A0A0J6K Histidine decarboxylase Chromobacterium sp. LK1 modified codon usage for A0A0J6KM89 M89 (HDC) (EC 4.1.1.22) Corynebacterium glutamicum and Saccharomyces cerevisiae 127 DNA seq for enzyme A0A0A1 Histidine decarboxylase Citrobacter pasteurii modified codon usage for A0A0A1R6V3 R6V3 (HDC) (EC 4.1.1.22) Corynebacterium glutamicum and Saccharomyces cerevisiae 128 DNA seq for enzyme A0A1W0 Histidine decarboxylase Chromobacterium haemolyticum modified codon usage for A0A1W0CM88 CM88 (HDC) (EC 4.1.1.22) Corynebacterium glutamicum and Histidine decarboxylase Saccharomyces cerevisiae 129 DNA seq6 for P00862 proenzyme Lactobacillus sp. (strain 30a) modified codon usage for enzyme P00862 (EC 4.1.1.22) (Pi chain) Corynebacterium glutamicum and [Cleaved into: Histidine Saccharomyces cerevisiae decarboxylase beta chain; Histidine decarboxylase alpha chain] 130 DNA seq for enzyme A0A0K6 Histidine decarboxylase Lactobacillus reuteri modified codon usage for A0A0K6GJ74 GJ74 proenzyme Corynebacterium glutamicum and Saccharomyces cerevisiae 131 DNA seq for P38620 Ribose-phosphate Saccharomyces cerevisiae modified codon usage for enzyme P38620 pyrophosphokinase 2 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (EC 2.7.6.1) (Phosphoribosyl (Baker's yeast) Saccharomyces cerevisiae pyrophosphate synthase 2) 132 DNA seq for P38689 Ribose-phosphate Saccharomyces cerevisiae modified codon usage for enzyme P38689 pyrophosphokinase 3 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (EC 2.7.6.1) (Phosphoribosyl (Baker's yeast) Saccharomyces cerevisiae pyrophosphate synthase 3) 133 DNA seq for P15019 Transaldolase (EC 2.2.1.2) Saccharomyces cerevisiae modified codon usage for enzyme P15019 (strain ATCC 204508/S288c) Corynebacterium glutamicum and (Baker's yeast) Saccharomyces cerevisiae 134 DNA seq for Q680A5 Ribose-phosphate Arabidopsis thaliana modified codon usage for enzyme Q680A5 pyrophosphokinase 4 (Mouse-ear cress) Corynebacterium glutamicum and (EC 2.7.6.1) (Phosphoribosyl Saccharomyces cerevisiae pyrophosphate synthase 4) 135 DNA seq for O59667 Histidine biosynthesis Schizosaccharomyces pombe modified codon usage for enzyme O59667 bifunctional (strain 972/ATCC 24843) Corynebacterium glutamicum and protein his7 [Includes: (Fission yeast) Saccharomyces cerevisiae Phosphoribosyl-AMP cyclohydrolase (EC 3.5.4.19); Phosphoribosyl-ATP pyrophosphatase (EC 3.6.1.31)] 136 DNA seq2 for P38635 Histidinol-phosphatase Saccharomyces cerevisiae modified codon usage for enzyme P38635 (HolPase) (EC 3.1.3.15) (strain ATCC 204508/S288c) Corynebacterium glutamicum and (Baker's yeast) Saccharomyces cerevisiae 137 DNA seq for A4QEF2 Glucose-6-phosphate Corynebacterium glutamicum modified codon usage for enzyme 1-dehydrogenase (strain R) Corynebacterium glutamicum and A4QEF2 with (G6PD) (EC 1.1.1.49) Saccharomyces cerevisiae substitution A243T 138 DNA seq7 for P00862 Histidine decarboxylase Lactobacillus sp. (strain 30a) Yarrowia lipolytica enzyme P00862 proenzyme 139 DNA seq5 A0A0J6K Histidine decarboxylase Chromobacterium sp. LK1 modified codon usage for for enzyme M89 Corynebacterium glutamicum and A0A0J6KM89 Saccharomyces cerevisiae 140 DNA seq6 Q9Z472 ATP phosphoribosyl transferase Corynebacterium glutamicum modified codon usage for for enzyme Q9Z472 ATCC 13032 Corynebacterium glutamicum and with substitution Saccharomyces cerevisiae N215K, L231F, T235A 141 DNA seq4 for Q9Z472 ATP phosphoribosyltransferase Corynebacterium glutamicum Saccharomyces cerevisiae enzyme Q9Z472 (strain ATCC 13032/ DSM 20300/JCM 1318/ LMG 3730/NCIMB 10025) 142 AA seq for N-terminal solubility tag 143 AA seq for E7EAU9 Ribose-phosphate Bacillus amyloliquefaciens Yarrowia lypolytica enzyme E7EAU9 pyrophosphokinase (RPPK)

REFERENCES

-   1. Gezginc, Y., et al., Biogenic amines formation in Streptococcus     thermophilus isolated from home-made natural yogurt. Food     Chem, 2013. 138(1): p. 655-62. -   2. Byun, B. Y. and J. H. Mah, Occurrence of biogenic amines in Miso,     Japanese traditional fermented soybean paste. J Food Sci, 2012.     77(12): p. T216-23. -   3. Landete, J. M., et al., Molecular methods for the detection of     biogenic amine-producing bacteria on foods. Int J Food     Microbiol, 2007. 117(3): p. 258-69. -   4. Ferstl, R., et al., Histamine receptor 2 is a key influence in     immune responses to intestinal histamine-secreting microbes. J     Allergy Clin Immunol, 2014. 134(3): p. 744-746 e3. -   5. Tabanelli, G., et al., Effect of chemico-physical parameters on     the histidine decarboxylase (HdcA) enzymatic activity in     Streptococcus thermophilus PRI60. J Food Sci, 2012. 77(4): p.     M231-7. -   6. Wauters, G., et al., Histidine decarboxylase in     Enterobacteriaceae revisited. J Clin Microbiol, 2004. 42(12): p.     5923-4. -   7. Lee, M. E., et al., A Highly Characterized Yeast Toolkit for     Modular, Multipart Assembly. ACS Synth Biol, 2015. 4(9): p. 975-86. -   8. Roland, B. P., et al., Triosephosphate isomerase I170V alters     catalytic site, enhances stability and induces pathology in a     Drosophila model of TPI deficiency. Biochim Biophys Acta, 2015.     1852(1): p. 61-9. 

What is claimed is:
 1. An engineered microbial cell that expresses a non-native histidine decarboxylase, wherein the engineered microbial cell produces histamine.
 2. The engineered microbial cell of claim 1, wherein the engineered microbial cell comprises increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell.
 3. The engineered microbial cell of claim 2, wherein the one or more upstream histamine pathway enzyme(s) are selected from the group consisting of an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase.
 4. The engineered microbial cell of claim 1, wherein the engineered microbial cell comprises reduced activity of one or more enzyme(s) that consume one or more histamine pathway precursors, said reduced activity being reduced relative to a control cell.
 5. The engineered microbial cell of claim 1, wherein the engineered microbial cell additionally expresses a feedback-deregulated glucose-6-phosphate dehydrogenase or a feedback-deregulated ATP phosphoribosyltransferase.
 6. An engineered microbial cell, wherein the engineered microbial cell comprises means for expressing a non-native histidine decarboxylase, wherein the engineered microbial cell produces histamine.
 7. The engineered microbial cell of claim 6, wherein the engineered microbial cell additionally comprises means for expressing glucose-6-phosphate dehydrogenase or a feedback-deregulated ATP phosphoribosyltransferase.
 8. The engineered microbial cell of claim 1, wherein the engineered microbial cell comprises a fungal cell.
 9. The engineered microbial cell of claim 1, wherein the non-native histidine decarboxylase comprises a histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 or from Acinetobacter baumannii strain AB0057.
 10. The engineered microbial cell of claim 1, wherein the engineered microbial cell is a bacterial cell.
 11. The engineered microbial cell of claim 1, wherein the engineered microbial cell comprises increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell, wherein the one or more upstream histamine pathway enzyme(s) comprise an ATP phosphoribosyltransferase and an imidazole-glycerol phosphate dehydratase.
 12. A culture of engineered microbial cells according to any one of claim
 1. 13. The culture of claim 12, wherein the culture comprises histamine at a level at least 20 mg/L of culture medium.
 14. A method of culturing engineered microbial cells according to claim 1, the method comprising culturing the cells under conditions suitable for producing histamine.
 15. A method for preparing histamine using microbial cells engineered to produce histamine, the method comprising: (a) expressing a non-native histidine decarboxylase in microbial cells; (b) cultivating the microbial cells in a suitable culture medium under conditions that permit the microbial cells to produce histamine, wherein the histamine is released into the culture medium; and (c) isolating histamine from the culture medium. 